(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
9 August 2001 (09.08.2001)
PCT
II
(10) International Publication Number
WO 01/57008 Al
(51) International Patent Classification 7 : C07D 277/82, (74) Agents: ELMORE, Carolyn, S. et aL; Hamilton, Brook,
417/04, 417/12, 417/06, 453/02, 451/02, 451/14, 513/04,
487/08, A61K 31/428, 31/435, 31/53, A61P 35/00
Smith & Reynolds, P.C., Two Militia Drive, Lexington, MA
02421 (US).
(21) International Application Number: PCT/US 01/03803
(22) International Filing Date: 6 February 2001 (06.02.2001)
(25) Filing Language:
(26) Publication Language:
English
English
(30) Priority Data:
60/180,841
7 February 2000 (07.02.2000) US
(71) Applicant (for all designated States except US): BASF
AKTIENGESELLSCHAFT [DE/DE]; Rheinland-Pfalz,
67056 Ludwigshafen (DE).
(72) Inventors; and
(75) Inventors/Applicants (for US only): CUSACK, Kevin,
P. [US/US]; 115 Jennifer Drive, Holden, MA 01520 (US).
SCOTT, Barbara [US/US]; 31 May Street, Spencer, MA
01520 (US). ARNOLD, Lee, D. [CA/US]; 216 Ruggles
Street, Weston, MA 01581 (US). ERICSSON, Anna
[SE/US]; 4133 Arbor Drive, Shrewsbury, MA 01545 (US).
(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
DE, DK, DM, DZ, EE, ES, FT, GB, GD, GE, GH, GM, HR,
HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ,
NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM,
TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FT, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
Published:
— with international search report
— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
<
(54) Title: 2-BENZOTHIAZOLYL UREA DERIVATIVES AND THEIR USE AS PROTEIN KINASE INHIBITORS
VCl
O
(I)
(57) Abstract: The present invention is directed to a compound of formula (I), racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutic ally -acceptable salts of said compound, isomers, prodrugs and
isotopes, wherein the variable are defined herein. The compounds of this invention are useful as inhibitors of serine/threonine and
tyrosine kinases. In particular, compounds of this invention are useful as inhibitors of tyrosine kinases that are important in hyper-
proliferative diseases, especially in cancer and in the process of angiogenesis.
WO 01/57008
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2-BENZOTHIAZOIjYL urea derivatives and their use as protein kinase inhibitors
BACKGROUND OF THE INVENTION
The present invention is directed to benzothiazole derivatives of formula (I), as defined
5 below, uses and pharmaceutical compositions thereof.
There are at least 400 enzymes identified as protein kinases. These enzymes catalyze the
phosphorylation of target protein substrates. The phosphorylation is usually a transfer reaction of a
phosphate group from ATP to the protein substrate. The specific structure in the target substrate to
which the phosphate is transferred is a tyrosine, serine or threonine residue. Since these amino acid
10 residues are the target structures for the phosphoryl transfer, these protein kinase enzymes are
commonly referred to as tyrosine kinases or serine/threonine kinases.
The phosphorylation reactions, and counteracting phosphatase reactions, at the tyrosine,
serine and threonine residues are involved in numerous cellular processes that underlie responses to
diverse intracellular signals (typically mediated through cellular receptors), regulation of cellular
15 functions, and activation or deactivation of cellular processes. A cascade of protein kinases often
participate in intracellular signal transduction and are necessary for the realization of these cellular
processes. Because of their ubiquity in these processes, the protein kinases can be found as an
integral part of the plasma membrane or as cytoplasmic enzymes or localized in the nucleus, often as
components of enzyme complexes. In many instances, these protein kinases are an essential element
20 of enzyme and structural protein complexes that determine where and when a cellular process occurs
within a cell.
Protein Tyrosine Kinases, Protein tyrosine kinases (PTKs) are enzymes which catalyze the
phosphorylation of specific tyrosine residues in cellular proteins. This post-translational
modification of these substrate proteins, often enzymes themselves, acts as a molecular switch
25 regulating cell proliferation, activation or differentiation (for review, see Schlessinger and Ulrich,
1992, Neuron 9:383-391). Aberrant or excessive PTK activity has been observed in many disease
states including benign and malignant proliferative disorders as well as diseases resulting from
inappropriate activation of the immune system (e.g., autoimmune disorders), allograft rejection, and
graft vs. host disease. In addition, endothelial-cell specific receptor PTKs such as KDR, Tie-2 and
30 Tie-1 mediate the angiogenic process, and are thus involved in supporting the progression of cancers
and other diseases involving inappropriate vascularization (e.g., diabetic retinopathy, choroidal
neovascularization due to age-related macular degeneration, psoriasis, arthritis, retinopathy of
prematurity, infantile hemangiomas).
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Tyrosine kinases can be of the receptor-type (having extracellular, transmembrane and
intracellular domains) or the non-receptor type (being wholly intracellular).
Receptor Tyrosine Kinases (RTKs). The RTKs comprise a large family of transmembrane
receptors with diverse biological activities. At present, at least nineteen (19) distinct RTK
5 subfamilies have been identified. The receptor tyrosine kinase (RTK) family includes receptors that
are crucial for the growth and differentiation of a variety of cell types (Yarden and Ullrich,^??/?. Rev,
Biochem. 57:433-478, 1988; Ullrich and Schlessinger, Cell 61:243-254, 1990). The intrinsic
function of RTKs is activated upon ligand binding, which results in phosphorylation of the receptor
and multiple cellular substrates, and subsequently in a variety of cellular responses (Ullrich &
10 Schlessinger, 1990, Cell 61:203-212). Thus, receptor tyrosine kinase mediated signal transduction is
initiated by extracellular interaction with a specific growth factor (ligand), typically followed by
receptor dimerization, stimulation of the intrinsic protein tyrosine kinase activity and receptor trans-
phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules
and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that
15 facilitate the appropriate cellular response, (e.g., cell division, differentiation, metabolic effects,
changes in the extracellular microenvironment) see Schlessinger and Ullrich, 1992, Neuron 9:1-20.
Proteins with SH2 (src homology-2) or phosphotyrosine binding (PTB) domains bind
activated tyrosine kinase receptors and their substrates with high affinity to propagate signals into
cell. Both of the domains recognize phosphotyrosine. (Fantl et al, 1992, Cell 69:413-423;
20 Songyang et al 9 1994, Mol Cell Biol 14:2777-2785; Songyang et al, 1993, Cell 72:767-778; and
Koch et aL, 1991, Science 252:668-678; Shoelson, Curr. Opin. Chem. Biol (1997), 1(2), 227-234;
Cowburn, Curr. Opin. Struct Biol (1997), 7(6), 835-838). Several intracellular substrate proteins
that associate with receptor tyrosine kinases (RTKs) have been identified. They may be divided into
two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack
25 such a domain but serve as adapters and associate with catalytically active molecules (Songyang
a/., 1993, Cell 12:761-17%). The specificity of the interactions between receptors or proteins and
SH2 or PTB domains of their substrates is determined by the amino acid residues immediately
surrounding the phosphorylated tyrosine residue. For example, differences in the binding affinities
between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on
30 particular receptors correlate with the observed differences in their substrate phosphorylation
profiles (Songyang et al., 1993, Cell 72:767-778). Observations suggest that the function of each
receptor tyrosine kinase is determined not only by its pattern of expression and ligand availability
but also by the array of downstream signal transduction pathways that are activated by a particular
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receptor as well as the timing and duration of those stimuli. Thus, phosphorylation provides an
important regulatory step which determines the selectivity of signaling pathways recruited by
specific growth factor receptors, as well as differentiation factor receptors.
Several receptor tyrosine kinases such as FGFR-1, PDGFR, Tie-2, Tie-1 and c-Met, and
5 growth factors that bind thereto, have been suggested to play a role in angiogenesis, although some
may promote angiogenesis indirectly (Mustonen and Alitalo, J. Cell Biol. 129:895-898, 1995). One
such receptor tyrosine kinase, known as "fetal liver kinase 1" (FLK-1), is a member of the type III
subclass of RTKs. An alternative designation for human FLK-1 is "kinase insert domain-containing
receptor" (KDR) (Terman et ah, Oncogene 6:1677-83, 1991). Another alternative designation for
10 FLK-1 /KDR is "vascular endothelial cell growth factor receptor 2" (VEGFR-2) since it binds VEGF
with high affinity. The murine version of FLK-1 /VEGFR-2 has also been called NYK (Oelrichse/
a/, Oncogene 8(1):1 1-15, 1993). DNAs encoding mouse, rat and human FLK-1 have been isolated,
and the nucleotide and encoded amino acid sequences reported (Matthews et ah, Proc. Natl Acad.
Sci. USA, 88:9026-30, 1991; Terman et al. y 1991, supra; Terman et al. 9 Biochem. Biophys. Res.
15 Comm. 187:1579-86, 1992; Sarzani et ah, supra; and Millauer et ah, Cell 72:835-846, 1993).
Numerous studies such as those reported in Millauer et ah, supra, suggest that VEGF and FLK-
l/KDR/VEGFR-2 are a ligand-receptor pair that play an important role in the proliferation of
vascular endothelial cells, and formation and sprouting of blood vessels, termed vasculogenesis and
angiogenesis, respectively.
20 Another type III subclass RTK designated "fms-like tyrosine kinase- 1" (Fit- 1 ) is related to
FLK-1 /KDR (DeVries et al. Science 255;989-991, 1992; Shibuya et al. ; Oncogene 5:519-524, 1990).
An alternative designation for Flt-1 is "vascular endothelial cell growth factor receptor 1" (VEGFR-
1). To date, members of the FLK-1/ KDR/VEGFR-2 and Flt-1/ VEGFR-1 subfamilies have been
found expressed primarily on endothelial cells. These subclass members are. specifically stimulated
25 by members of the vascular endothelial cell growth factor (VEGF) family of ligands (Klagsburn and
D'Amore, Cytokine & Growth Factor Reviews 7: 259-270, 1996). Vascular endothelial cell growth
factor (VEGF) binds to Flt-1 with higher affinity than to FLK-1 /KDR and is mitogenic toward
vascular endothelial cells (Terman et al., \992, supra; Mustonen et al. supra; DeVries et al., supra).
Flt-1 is believed to be essential for endothelial organization during vascular development. Flt-1
30 expression is associated with early vascular development in mouse embryos, and with
neovascularization during wound healing (Mustonen and Alitalo, supra). Expression of Flt-1 in
monocytes, osteoclasts, and osteoblasts, as well as in adult tissues such as kidney glomeruli suggests
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an additional function for this receptor that is not related to cell growth (Mustonen and Alitalo,
supra).
As previously stated, recent evidence suggests that VEGF plays a role in the stimulation of
both normal and pathological angiogenesis (Jakeman et al., Endocrinology 133: 848-859, 1993;
5 Kolch et al., Breast Cancer Research and Treatment 36: 139-155, 1995; Ferrara et a!., Endocrine
Reviews 18(1); 4-25, 1997; Ferrara et al., Regulation of Angiogenesis (ed. L. D. Goldberg and E.M.
Rosen), 209-232, 1997). In addition, VEGF has been implicated in the control and enhancement of
vascular permeability (Connolly, et al., J. Biol Chem. 264: 20017-20024, 1989; Brown et al,
Regulation of Angiogenesis (ed. L.D. Goldberg and E.M. Rosen), 233-269, 1997). Different forms of
10 VEGF arising from alternative splicing of mRNA have been reported, including the four species
described by Ferrara et al (J. Cell. Biochem. 47:21 1-218, 1991). Both secreted and predominantly
cell-associated species of VEGF have been identified by Ferrara et al supra, and the protein is
known to exist in the form of disulfide linked dimers.
Several related homologs of VEGF have recently been identified. However, their roles in
15 normal physiological and disease processes have not yet been elucidated. In addition, the members
of the VEGF family are often coexpressed with VEGF in a number of tissues and are, in general,
capable of forming heterodimers with VEGF. This property likely alters the receptor specificity and
biological effects of the heterodimers and further complicates the elucidation of their specific
functions as illustrated below (Korpelainen and Alitalo, Curr. Opin. Cell Biol, 159-164, 1998 and
20 references cited therein).
Placenta growth factor (P1GF) has an amino acid sequence that exhibits significant
homology to the VEGF sequence (Park et al, J. Biol. Chem. 269:25646-54, 1994; Maglione et al.
Oncogene 8:925-31, 1993). As with VEGF, different species of P1GF arise from alternative splicing
of mRNA, and the protein exists in dimeric form (Parked aL, supra). P1GF-1 and P1GF-2 bind to
25 Flt-1 with high affinity, and P1GF-2 also avidly binds to neuropilin-1 (Migdal^ al, J. Biol. Chem.
273 (35): 22272-22278), but neither binds to FLK-1/KDR (Park et al, supra). P1GF has been
reported to potentiate both the vascular permeability and mitogenic effect of VEGF on endothelial
cells when VEGF is present at low concentrations (purportedly due to heterodimer formation) (Park
et al., supra).
30 VEGF-B is produced as two isoforms (167 and 185 residues) that also appear to bind Flt-
1/VEGFR-l . It may play a role in the regulation of extracellular matrix degradation, cell adhesion,
and migration through modulation of the expression and activity of urokinase type plasminogen
4
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activator and plasminogen activator inhibitor 1 (Pepper^ al, Proc. Natl Acad. ScL U. S. A. (1998),
95(20): 11709-11714).
VEGF-C was originally cloned as a Iigand for VEGFR~3/Flt-4 which is primarily expressed
by lymphatic endothelial cells. In its fully processed form, VEGF-C can also bind KDR/VEGFR-2
5 and stimulate proliferation and migration of endothelial cells in vitro and angiogenesis in in vivo
models ( Lymboussaki et al 9 Am, J. Pathol (1998), 153(2): 395-403; Witzenbichler et al 9 Am. J.
Pathol (1998), 153(2), 381-394). The transgenic overexpression of VEGF-C causes proliferation
and enlargement of only lymphatic vessels, while blood vessels are unaffected. Unlike VEGF, the
expression of VEGF-C is not induced by hypoxia (Ristimaki et al 9 J. Biol Chem. (1998),
10 273(14),8413~841S).
The most recently discovered VEGF-D is structurally very similar to VEGF-C. VEGF-D is
reported to bind and activate at least two VEGFRs, VEGFR-3/Flt-4 and KDR/VEGFR-2. It was
originally cloned as a c-fos inducible mitogen for fibroblasts and is most prominently expressed in
the mesenchymal cells of the lung and skin (Achen et al 9 Proc. Natl Acad. Sci. U. S. A. (1998),
15 95(2), 548-553 and references therein).
As for VEGF, VEGF-C and VEGF-D have been claimed to induce increases in vascular
permeability in vivo in a Miles assay when injected into cutaneous tissue (PCT/US97/14696;
WO98/07832, Witzenbichler et al. 9 supra). The physiological role and significance of these ligands
in modulating vascular hyperpermeability and endothelial responses in tissues where they are
20 expressed remains uncertain.
There has been recently reported a viral ly encoded, novel type of vascular endothelial
growth factor, VEGF-E (NZ-7 VEGF), which preferentially utilizes KDR/Flk-1 receptor and carries
a potent mitotic activity without heparin-binding domain (Meyere/ a/, EMBOJ. (1999), 18(2), 363-
374; Ogawa et al 9 J. Biol Chem. (1998), 273(47), 31273-31282.). VEGF-E sequences possess 25%
25 homology to mammalian VEGF and are encoded by the parapoxvirus Orf virus (OV). This
parapoxvirus that affects sheep and goats and occasionally, humans, to generate lesions with
angiogenesis. VEGF-E is a dimer of about 20 kDa with no basic domain nor affinity for heparin, but
has the characteristic cysteine knot motif present in all mammalian VEGFs, and was surprisingly
found to possess potency and bioactivities similar to the heparin-binding VEGF 165 isoform of
30 VEGF-A, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis
and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like
VEGF165, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in
5
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receptor autophosphorylation and a biphasic rise in free intracellular Ca^+ concentrations, while in
contrast to VEGF 165, VEGF-E did not bind to VEGF receptor- 1 (Flt-1).
Based upon emerging discoveries of other homologs of VEGF and VEGFRs and the
precedents for ligand and receptor heterodimerization, the actions of such VEGF homologs may
5 involve formation of VEGF ligand heterodimers, and/or heterodimerization of receptors, or binding
to a yet undiscovered VEGFR (Witzenbichler et aL, supra). Also, recent reports suggest neuropilin-
1 (Migdal et al, supra) or VEGFR-3/Flt-4 (Witzenbichler et aL, supra), or receptors other than
KDR/VEGFR-2 may be involved in the induction of vascular permeability (Stacker, S.A., Vitali, A. 5
Domagala, T., Nice, E., and Wilks, A.F., Angiogenesis and Cancer Conference, Amer. Assoc.
10 Cancer Res., Jan. 1998, Orlando, FL; Williams, Diabetelogia 40: SI 18-120 (1997)).
Tie-2 (TEK) is a member of a recently discovered family of endothelial cell specific receptor
tyrosine kinases which is involved in critical angiogenic processes, such as vessel branching,
sprouting, remodeling, maturation and stability. Tie-2 is the first mammalian receptor tyrosine
kinase for which both agonist ligand(s) (e.g., Angiopoietinl ("Angl"), which stimulates receptor
15 autophosphorylation and signal transduction), and antagonist ligand(s) (e.g., Angiopoietin2
("Ang2")), have been identified. Knock-out and transgenic manipulation of the expression of Tie-2
and its ligands indicates tight spatial and temporal control of Tie-2 signaling is essential for the
proper development of new vasculature. The current model suggests that stimulation of Tie-2 kinase
by the Angl ligand is directly involved in the branching, sprouting and outgrowth of new vessels,
20 and recruitment and interaction of periendothelial support cells important in maintaining vessel
integrity and inducing quiescence. The absence of Angl stimulation of Tie-2 or the inhibition of
Tie-2 autophosphorylation by Ang2, which is produced at high levels at sites of vascular regression,
may cause a loss in vascular structure and matrix contacts resulting in endothelial cell death,
especially in the absence of growth/survival stimuli. The situation is however more complex, since
25 at least two additional Tie-2 ligands (Ang3 and Ang4) have recently been reported, and the capacity
for heterooligomerization of the various agonistic and antagonistic angiopoietins, thereby modifying
their activity, has been demonstrated. Targeting Tie-2 ligand-receptor interactions as an
antiangiogenic therapeutic approach is thus less favored and a kinase inhibitory strategy preferred.
The soluble extracellular domain of Tie-2 ("ExTek") can act to disrupt the establishment of
30 tumor vasculature in a breast tumor xenograft and lung metastasis models and in tumor-cell
mediated ocular neovasculatization. By adenoviral infection, the/>? vivo production of mg/ml levels
ExTek in rodents may be achieved for 7-10 days with no adverse side effects. These results suggest
that disruption of Tie-2 signaling pathways in normal healthy animals may be well tolerated. These
6
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Tie-2 inhibitory responses to ExTek may be a consequence sequestration of ligand(s) and/or
generation of a non-productive heterodimer with full-length Tie-2.
Recently, significant upregulation of Tie-2 expression has been found within the vascular
synovial pannus of arthritic joints of humans, consistent with a role in the inappropriate
5 neovascularization. This finding suggests that Tie-2 plays a role in the progression of rheumatoid
arthritis. Point mutations producing constitutively activated forms of Tie-2 have been identified in
association with human venous malformation disorders. Tie-2 inhibitors are, therefore, useful in
treating such disorders, and in other situations of inappropriate neovascularization.
The Non-Receptor Tyrosine Kinases. The non-receptor tyrosine kinases represent a
10 collection of cellular enzymes which lack extracellular and transmembrane sequences. At present,
over twenty-four individual non-receptor tyrosine kinases, comprising eleven (1 1) subfamilies (Src,
Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack and LIMK) have been identified. At present, the
Src subfamily of non-receptor tyrosine kinases is comprised of the largest number of PTKs and
include Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. The Src subfamily of enzymes has been
15 linked to oncogenesis and immune responses. A more detailed discussion of non-receptor tyrosine
kinases is provided in Bolen, 1993, Oncogene 8:2025-2031, which is incorporated herein by
reference.
Many of the tyrosine kinases, whether an RTK or non-receptor tyrosine kinase, have been
found to be involved in cellular signaling pathways involved in numerous pathogenic conditions,
20 including cancer, psoriasis, and other hyperproliferative disorders or hyper-immune responses.
Development of Compounds to Modulate the PTKs. In view of the surmised importance of
PTKs to the control, regulation, and modulation of cell proliferation, the diseases and disorders
associated with abnormal cell proliferation, many attempts have been made to identify receptor and
non-receptor tyrosine kinase "inhibitors" using a variety of approaches, including the use of mutant
25 ligands (U.S. Application No. 4,966,849), soluble receptors and antibodies (Application No. WO
94/10202; Kendall & Thomas, \994, Proc. Natl Acad. Sci 90:10705-09; Kim et ah, 1993, Nature
362:841-844), RNA ligands (Jellinek, et aL, Biochemistry 33:10450-56; Takano, et ah, 1993, Mol
Bio. Cell 4:358A; Kinsella, et aL 1992, Exp. Cell Res. 199:56-62; Wright, et aL, 1992, J. Cellular
Phys. 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO
30 94/14808; U.S. Patent No. 5,330,992; Mariani, et aL, 1994, Proc. Am. Assoc. Cancer Res. 35:2268).
More recently, attempts have been made to identify small molecules which act as tyrosine
kinase inhibitors. For example, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO
92/20642) and vinylene-azaindole derivatives (PCT WO 94/14808) have been described generally as
7
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tyrosine kinase inhibitors. Styryl compounds (U.S. Patent No. 5,217,999), styryl-substituted pyridyl
compounds (U.S. Patent No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566
266 Al; Expert Opin. Ther. Pat (1998), 8(4): 475-478), selenoindoles and selenides (PCT WO
94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid
5 compounds (PCT WO 91/15495) have been described as compounds for use as tyrosine kinase
inhibitors for use in the treatment of cancer. Anilinocinnolines (PCT W097/34876) and quinazoline
derivative compounds (PCT W097/22596; PCT W097/42187) have been described as inhibitors of
angiogenesis and vascular permeability.
In addition, attempts have been made to identify small molecules which act as
10 serine/threonine kinase inhibitors. For example, bis(indolylmaleimide) compounds have been
described as inhibiting particular PKC serine/threonine kinase isoforms whose signal transducing
function is associated with altered vascular permeability in VEGF-related diseases (PCT
WO97/40830; PCT WO97/4083 1).
Plk-1 Kinase Inhibitors
15 pik-1 is a serine/threonine kinase which is an important regulator of cell cycle progression.
It plays critical roles in the assembly and the dynamic function of the mitotic spindle apparatus. Plk-
1 and related kinases have also been shown to be closely involved in the activation and inactivation
of other cell cycle regulators, such as cyclin-dependent kinases. High levels of Plk-1 expression are
associated with cell proliferation activities. It is often found in malignant tumors of various origins.
20 Inhibitors of Plk-1 are expected to block cancer cell proliferation by disrupting processes involving
mitotic spindles and inappropriately activated cyclin-dependent kinases.
Cdc2/Cyclin B Kinase Inhibitors (Cdc2 is also known as cdkl)
Cdc2/cyclin B^is another serine/threonine kinase enzyme which belongs to the cyclin-
dependent kinase (cdk) family. These enzymes are involved in the critical transition between
25 various phases of cell cycle progression. It is believed that uncontrolled cell proliferation, which is
the hallmark of cancer is dependent upon elevated cdk activities in these cells. The inhibition of
elevated cdk activities in cancer cells by cdc2/cyclin B kinase inhibitors can suppress proliferation
and may restore the normal control of cell cycle progression.
The regulation of CDK activation is complex, but requires the association of the CDK with a
30 member of the cyclin family of regulatory subunits (Draetta, Trends in Cell Biology, 3:287-289
(1993)); Murray and Kirschner, Nature, 339:275-280 (1989); Solomon et al, Molecular Biology of
the Cell, 3:13-27 (1992)). A further level of regulation occurs through both activating and
inactivating phosphorylations of the CDK subunit (Draetta, Trends in Cell Biology, 3:287-289
8
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PCT/US01/03803
(1993)); Murray and Kirschner, Nature, 339:275-280 (1989); Solomon et aL, Molecular Biology of
the Cell, 3:13-27 (1992); Ducommun et aL, EMBO Journal, 10:3311-3319 (1991); Gautier et aL,
Nature 339:626-629 (1989); Gould and Nurse, Nature, 342:39-45 (1989); Krek and Nigg, EMBO
Journal 10:3331-3341 (1991); Solomon et aL, Cell, 63:1013-1024 (1990)). The coordinate
5 activation and inactivation of different cyclin/CDK complexes is necessary for normal progression
through the cell cycle (Pines, Trends in Biochemical Sciences, 18:195-197 (1993); Sherr, Cell,
73:1059-1065 (1993)). Both the critical Gl-S and G2-M transitions are controlled by the activation
of different cyclin/CDK activities. In Gl, both cyclin D/CDK4 and cyclin E/CDK2 are thought to
mediate the onset of S-phase (Matsushima et aL, Molecular & Cellular Biology, 14:2066-2076
10 (1994); Ohtsubo and Roberts, Science, 259:1908-1912 (1993); Quelle et aL, Genes & Development,
7:1559-1571 (1993); Resnitzky et aL, Molecular & Cellular Biology, 14:1669-1679 (1994)).
Progression through S-phase requires the activity of cyclin A/CDK2 (Girard et aL, Cell, 67:1169-
1 179 (1991); Pagano et aL, EMBO Journal, 11:961-971 (1992); Rosenblatt et aL, Proceedings of the
National Academy of Science USA, 89:2824-2828 (1992); Walker and Mailer, Nature, 354:314-317
15 (1991); Zindy et aL, Biochemical & Biophysical Research Communications, 182:1 144-1 154 (1992))
whereas the activation of cyclin A/cdc2 (CDK1) and cyclin B/cdc2 are required for the onset of
metaphase (Draetta, Trends in Cell Biology, 3:287-289 (1993)); Murray and Kirschner, Nature,
339:275-280 (1989); Solomon et aL, Molecular Biology of the Cell, 3:13-27 (1992); Girard et aL,
Cell, 67:1169-1179 (1991); Pagano et aL, EMBO Journal, 11:961-971 (1992); Rosenblatt et aL,
20 Proceedings of the National Academy of Science USA, 89:2824-2828 (1992); Walker and Mailer,
Nature, 354:314-317 (1991); Zindy et aL, Biochemical & Biophysical Research Communications,
182:1 144-1 154 (1992)). It is not surprising, therefore, that the loss of control of CDK regulation is a
frequent event in hyperproliferative diseases and cancer. (Pines, Current Opinion in Cell Biology,
4:144-148 (1992); Lees, Current Opinion in Cell Biology,7:713-J$0 (1995); Hunter and Pines, Cell,
25 79:573-582 (1994)).
Inhibitors of kinases involved in mediating or maintaining disease states represent novel
therapies for these disorders. Examples of such kinases include, but are not limited to: (1) inhibition
of c-Src (Brickell, Critical Reviews in Oncogenesis, 3:401-406 (1992); Courtneidge, Seminars in
Cancer Biology, 5:236-246 (1994), raf (Powis, Pharmacology & Therapeutics, 62:57-95 (1994)) and
30 the cyclin-dependent kinases (CDKs) 1, 2 and 4 in cancer (Pines, Current Opinion in Cell Biology,
4;144_148 (1992); Lees, Current Opinion in Cell Biology,! ':773-780 (1995); Hunter and Pines, Cell,
79:573-582 (1994)), (2) inhibition of CDK2 or PDGF-R kinase in restenosis (Buchdunger et aL,
Proceedings of the National Academy of Science USA, 92:2258-2262 (1995)), (3) inhibition of
9
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CDK5 and GSK3 kinases in Alzheimers (Hosoi et al, Journal of Biochemistiy (Tokyo), 117:741-749
(1995); Aplin et al, Journal of Neurochemistry, 67:699-707 (1996), (4) inhibition of c-Src kinase in
osteoporosis (Tanaka et al, Nature, 383:528-531 (1996), (5) inhibition of GSK-3 kinase in type-2
diabetes (Borthwick et al, Biochemical & Biophysical Research Communications, 210:738-745
5 (1995), (6) inhibition of the p38 kinase in inflammation (Badger et al, The Journal of Pharmacology
and Experimental Therapeutics, 279:1453-1461 (1996)), (7) inhibition of VEGF-R 1-3 and TIE-1
and -2 kinases in diseases which involve angiogenesis (Shawver et ah, Drug Discovery Today, 2:50-
63 (1997)), (8) inhibition of UL97 kinase in viral infections (Heet al, Journal of Virology, 71:405-
411 (1997)), (9) inhibition of CSF-1R kinase in bone and hematopoetic diseases (Myers et al,
10 Bioorganic & Medicinal Chemistry Letters, 7:421-424 (1997), and (10) inhibition of Lck kinase in
autoimmune diseases and transplant rejection (Myers et ah, Bioorganic & Medicinal Chemistry
Letters, 7:417-420 (1997)).
It is additionally possible that inhibitors of certain kinases may have utility in the treatment
of diseases when the kinase is not misregulated, but it nonetheless essential for maintenance of the
15 disease state. In this case, inhibition of the kinase activity would act either as a cure or palliative for
these diseases. For example, many viruses, such as human papilloma virus, disrupt the cell cycle
and drive cells into the S-phase of the cell cycle (Vousden, FASEB Journal, 7:8720879 (1993)).
Preventing cells from entering DNA synthesis after viral infection by inhibition of essential S-phase
initiating activities such as CDK2, may disrupt the virus life cycle by preventing virus replication.
20 This same principle may be used to protect normal cells of the body from toxicity of cycle-specific
chemotherapeutic agents (Stone et al, Cancer Research, 56:3199-3202 (1996); Kohn et al, Journal
of Cellular Biochemistry, 54:44-452 (1994)). Inhibition of CDKs 2 or 4 will prevent progression
into the cycle in normal cells and limit the toxicity of cytotoxics which act in S-phase, G2 or mitosis.
Furthermore, CDK2/cyclin E activity has also been shown to regulate NF-kB. Inhibition of CDK2
25 activity stimulates NF-kB-dependent gene expression, an event mediated through interactions with
the p300 coactivator (Perkins et al, Science, 275:523-527 (1997)). NF-kB regulates genes involved
in inflammatory responses (such as hematopoetic growth factors, chemokines and leukocyte
adhesion molecules) (Baeuerle and Henkel, Annual Review of Immunology, 12:141-179 (1994)) and
may be involved in the suppression of apoptotic signals within the cell (Beg and Baltimore,£c/ewce,
30 274:782-784 (1996); Wang et al, Science, 274:784-787 (1996); Van Antwerp et al, Science,
274:787-789 (1996)). Thus, inhibition of CDK2 may suppress apoptosis induced by cytotoxic drugs
via a mechanism which involves NF-kB. This therefore suggests that inhibition of CDK2 activity
may also have utility in other cases where regulation of NF-kB plays a role in etiology of disease. A
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10
15
20
further example may be take from fungal infections: Aspergillosis is a common infection in
immune-compromised patients (Armstrong, Clinical Infectious Diseases, 16:1-7 (1993)). Inhibition
of the Aspergillus kinases Cdc2/CDC28 or Nim A (Osmani et al, EMBO Journal, 10:2669-2679
(1991); Osmani et al. 9 Cell, 67:283-291 (1991)) may cause arrest or death in the fungi, improving
the therapeutic outcome for patients with these infections.
The identification of effective small compounds which specifically inhibit signal
transduction and/or cellular proliferation by modulating the activity of receptor and non-receptor
tyrosine and serine/threonine kinases to regulate and modulate abnormal or inappropriate cell
proliferation, differentiation, or metabolism is therefore desirable. In particular, the identification of
methods and compounds that specifically inhibit the function of a tyrosine kinase which is essential
for angiogenic processes or the formation of vascular hyperpermeability leading to edema, ascites,
effusions, exudates, macromolecular extravasation and matrix deposition as well as associated
disorders would be beneficial.
SUMMARY OF THE INVENTION
In one aspect, the present invention is directed to a compound of formula (I),
N — R
(I).
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein,
Q is H or represents a bond which is taken together with X 1 and the two nitrogen atoms to which Q
and Xl are attached and the OY group to which the two nitrogen atoms are attached to form
Y
N
N — R
N
Q
Ql is (Ci-C 6 )alkyl;
Y is O or S;
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W is H, CI, Br, I, N0 2 , CN, SCN, OCF 3 , -Xq-CCCRlO^-Ylq-CCCR 1 °)2) a -Z 1 q . or an optionally
substituted group selected from the group consisting of alkyl, alkenyl, alkynyl, heterocyclyl-alkenyl,
and heterocyclyl-alkynyl;
Y 1 and X are each independently selected from the group consisting of phenyl, heterocyclyl,
NRl°, O, S, SO, S0 2 , CF 2 , CFR, C=0, (C=O)NRl0, SONRlO, SO 2 NRl0, NR™(C=0),
NR 10 SO,
R 10
N
J?
NR* OS0 2: NRl 0sO 2 NRl 0, NRl 0(C=O)NR 1 ° ;
O
O o 0
R
I
N
10
R
I
N
10
R
I
N
10
O 0
and
o 0
q for each occurrence is independently 0 or 1 ;
10 a for each occurrence is independently 0 or an integer from 1 to 5;
RlO f or each occurrence is independently selected from the group consisting of H ?
optionally substituted aryl, optionally substituted heterocyclyl and an optionally substituted
alkyl group optionally substituted with one or more of the following: a Ci_6 alkyl group
optionally substituted by one or more hydroxy, halo or optionally substituted amino; a C]_6
15 alkoxy group optionally substituted by one or more hydroxy, halo or optionally substituted
amino; hydroxy; halo; or optionally substituted amino;
Z 1 is H, optionally substituted alkyl, optionally substituted aryl or optionally substituted
heterocyclyl;
<■>
X 1 is hydrogen, alkyl, hydroxyalkyl or represents a bond which is taken together with RP as
20 described below or represents a bond which is taken together with Q as described above;
Rl and R 2 are each independently hydrogen, halogen, hydroxy, nitro, cyano, COOH, COOX 3 , SX J ,
S0 2 X3 5 SOX3, C(0)X3, NHC(0)X3, C(0)NHX3, NHS0 2 X3 G r selected from an optionally
substituted group consisting of alkyl, alkenyl, alkynyl, alkoxy, amino, NHX3, NX3x3, alkylamino,
arylamino, heterocyclylamino, alkylthio, alkylsulfonato, aryl, aryloxy, arylalkyl, arylalkenyl,
25 arylalkynyl, arylalkyloxy, heterocyclyl, heterocyclyloxy, heterocyclyl-alkyl, heterocyclyl-alkenyl,
12
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heterocyclyl-alkynyl, heterocyclyl-alkyloxy, heterocyclylthio, heterocyclylsulfinyl,
heterocyclylsulfonyl, cycioalkyl, -(CH 2 ) m -(CHX2)CN, -(CH 2 ) m -(CHX2)COOH, -(CH 2 )m-
(CHX2)COOX3 ? -(CH 2 )ni-(CHX2)S02X3, -(CH 2 ) m -(CHX2)C(0)X3, -(CH 2 ) m -
(CHX2)C(0)NHX3 and
5 -(CH 2 ) m -(CHX 2 )NHS0 2 X 3 ;
where m is 0 to 4;
X 2 for each occurrence is independently H or an optionally substituted moiety selected from
the group consisting of alkyl, alkenyl, alkynyl, carbonyl, S(0) p alkyl, S(0) p aryl,
S(0)pheterocyclyl, amino, alkoxy, alkylthio, arylthio, perhaloalkyl, aryl, aryloxy, arylalkyi,
10 arylalkyloxy, heterocyclyl and heterocyclyl-alkyl;
p is 0, 1 or 2;
X 3 for each occurrence is independently H or an optionally substituted moiety selected from
the group consisting of mono- or di-alkylamino, alkyl, alkenyl, alkynyl, aryl, arylalkyi,
heterocyclyl and heterocyclyl-alkyl;
15 or when is in the 7-position of the benzothiazole ring, Rj and W can be taken together with the
carbon atoms to which they are attached to form an optionally substituted 5- or 6-membered
heterocyclyl ring;
R3 is hydrogen, or an optionally substituted moiety selected from the group consisting of carbonyl,
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyi, heterocyclyl, heterocyclyl-alkyl, heterocyclyl-
20 heterocyclyl, heterocyclyl-cycloalkyl, amino, alkylamino, arylamino, alkoxy, thioalkoxy and acyl;
or R3 and are taken together with the nitrogen atom to which they are attached to form
1N or \^
*
where Z for each occurrence is independently selected from the group consisting of oxo, or
an optionally substituted moiety selected from the group consisting of-C(0)(C]-C6)alkyI,
25 -C(0)ary 1, -C(0)N(C ] -C 6 )alky 1, -C(0)N-ary 1, (C j -C 6 )alky 1, (C 2 -C 6 )alkeny 1, (C 2 -
C^alkynyl, amino, mono- or di-(Ci-C6)alkylamino, -COO(C]-C6)alkyl, pyridyl, phenyl,
phenyl(Ci-Cg)alkyl and phenyl(C]-Cg)alkenyl;
where each of the optionally substituted moieties described hereinabove is optionally substituted by
one or more substituents each independently selected from the group consisting of oxo, amino, nitro,
30 mono- or bi-(Ci-C6)alkylamino, hydroxy, nitrile, chloro, fluoro, bromo, iodo, CF3, (Ci-C6)alkyl,
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-C(0)(Ci-C 6 )alkyl, -COOH, -COO(C j-C 6 )aIkyl, -S~(C]-C 6 )alkyl, -S-aryl, (Ci-C 6 )alkoxy, -
SO2NH2, phenyl, phenyl(Ci-C6)alkyl, -0-(C]-C 6 )alkyl-OH, -0-(Ci-C6)alkyl-0-(C 1 -C6)alkyl, -O-
(C 2 -C 6 )alkyl-N-((C 1 -C 6 )alky l) n , -N-(C ] -C 6 )alkyl-OH, -N-(C 1 -C 6 )alkyl-0-(C 1 -C 6 )alky 1, -
C(0)NH 2 , -C(0)N((C]-C 6 )aIkyl) n , ,-S(0) n (Ci-C 6 )aIkyl, -S(0) n aiyl, -S(0) n heterocyclyl, and
5 heterocyclyl, where the alkyl groups mentioned herein optionally have one or more unsaturated
bonds in the alkyl portion;
n is 0, 1 or 2;
provided that
1) when Q is H; Y is O; R 1 and R 2 are each hydrogen, halogen, alkyl, alkoxy, alkylthio,
10 carboxyalkyl or optionally substituted phenyl; and X 1 is hydrogen or alkyl; then R-^ is not alkyl,
alkenyl, alkoxy, cycloalkyl or optionally substituted phenyl;
2) when Q is H; Y is O; R 1 and R 2 are each hydrogen, halogen, alkyl, alkoxy, alkylthio,
carboxyalkyl or optionally substituted phenyl; then X* and R 3 are not taken together to form
optionally substituted by 1 to 3 substituents independently selected from the group consisting of
amino, mono- or bi-(C]-C6)alkylamino, hydroxy, chloro, fluoro, bromo, iodo, (Ci-C6)alkyl, (Cj-
C6)alkoxy and -SO2NH2;
4) when W is CI, Br or I; Q is H; R 1 is 7-CI; R 2 is H; and X 1 is alkyl; then R 3 is not alkyl, alkoxy or
20 cycloalkyl;
5) when W is CI, Br or I; Q is H; R 1 is 7-CI; R 2 is H; and X* is H; then R 3 is not alkyl or
cycloalkylamino;
6) when W is CI, Br, I or NO2; Q is H; Y is O; X 1 is H; R 1 is OH; R 2 is NO2, amino, alkyl, alkoxy,
hydroxy lower alkyl or dialkylamino; then R 3 is not H or alkyl;
25 7) when W is CI, Br or I; Q is H; Y is O; R 1 is CF3, CH2F, NO2, alkyl or alkoxy; R 2 is H; X* is H;
then R 3 is not naphthyl or phenyl optionally substituted with halo, CF3, alkyl or alkoxy;
8) when W is CI, Br or I; Q is H; R 1 is alkyl; R 2 is H; X 1 is H or alkyl; then R 3 is not alkyl or
alkoxy;
9) when W is CI; Q is H; Y is S; R 1 and R 2 are each H; X 1 is H; then R 3 is not ethyl;
15 3) when W is CI, Br or I; Q is hydrogen; Y is O; X 1 is H; then R 3 is not
O
or phenyl
14
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10) when W is CI; Q is H; Y is O; R 1 and R 2 are each H; X 1 is H; then R 3 is not n-butyl; and
1 1 ) when W is H, then R 1 and R 2 are not H at the same time.
In a preferred embodiment, the present invention is directed to compounds of the formula
,1
S
Q
t
N
X 1
I ■
N — R
Y
10
15
20
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein,
Q is H or represents a bond which is taken together with X 1 and the two nitrogen atoms to which Q
and X 1 are attached and the OY group to which the two nitrogen atoms are attached to form
Y
N
N —
N
25
Q
Ql is(Ci-C6)alkyl;
Y is O or S;
W is CI, Br, I, N0 2 or CN;
w here xl is hydrogen, alkyl, hydroxyalkyl or represents a bond which is taken together with
r3 as described below or represents a bond which is taken together with Q as described
above;
Rl and R 2 are each independently hydrogen, halogen, hydroxy, nitro, cyano, COOH, COOX 3 ,
SCbX 3 , SOX 3 , C(0)X 3 , NHC(0)X 3 ? C(G)NHX 3 , NHS0 2 X 3 or selected from an optionally
substituted group consisting of alkyl, alkenyl, alkynyl, alkoxy, amino, NHX 3 , NX^X 3 , alkylamino,
arylamino, heterocyclylamino, alkylthio, alkylsulfonato, aryl, aryloxy, arylalkyl, arylalkenyl,
arylalkynyl, arylalkyloxy, heterocyclyl, heterocyclyloxy, heterocyclyl-alkyl, heterocyclyl-alkenyl,
heterocyclyl-alkynyl, heterocyclyl-alkyloxy, heterocyclylthio, heterocyclylsulfinyl,
heterocyclylsulfonyl, cycloalkyl, -(CH 2 ) m -(CHX 2 )CN, -(CH 2 ) m -(CHX 2 )COOH, -(CH 2 ) m -
(CHX 2 )COOX 3 , -(CH 2 ) m -(CHX 2 )S0 2 X 3 , -(CH 2 ) m -(CHX 2 )C(0)X 3 , -(CH 2 ) m -
(CHX 2 )C(0)NHX 3 and
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-(CH2) m -(CHX2)NHSC>2X3 ;
where m is 0 to 4;
X 2 for each occurrence is independently H or an optionally substituted moiety selected from
the group consisting of alkyl, alkenyl, alkynyl, carbonyl, S(0) p alkyl, S(0) p aryl,
5 S(0) p heterocyclyl, amino, alkoxy, alkylthio, arylthio, perhaloalkyl, aryl, aryloxy, arylalkyl,
arylalkyloxy, heterocyclyl and heterocyclyl-alkyl;
p is 0, 1 or 2;
X 3 for each occurrence is independently H or an optionally substituted moiety selected from
the group consisting of mono- or di-alkylamino, alkyl, alkenyl, alkynyl, aryl, arylalkyl,
10 heterocyclyl and heterocyclyl-alkyl;
R 3 is hydrogen, or an optionally substituted moiety selected from the group consisting of carbonyl,
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heterocyclyl, heterocyclyl-alkyl, amino,
alkylamino, arylamino, alkoxy, thioalkoxy and acyl;
or R 3 and X 1 are taken together with the nitrogen atom to which they are attached to form
15 ;
where Z for each occurrence is independently selected from the group consisting of oxo, or
an optionally substituted moiety selected from the group consisting of-C(0)(C]-C6)alkyl,
-C(0)ary 1, -C(0)N(C i -C6)alky 1, -C(0)N-aryl, (C i -C 6 )alky I, (C 2 -C6)alkeny 1, (C 2 -
C6)alkynyl, amino, mono- or di-(Ci-C6)alkylamino, -COO(Ci-C6)alkyl, pyridyl, phenyl,
20 phenyl(C ] -C6)alkyl and phenyl(C ] -C6)alkenyl;
where each of the optionally substituted moieties described hereinabove is optionally substituted by
one or more substituents each independently selected from the group consisting of oxo, amino, nitro,
mono- or bi-(Ci-C6)alkylamino, hydroxy, nitrile, chloro, fluoro, bromo, iodo, CF3, (C j~C6)alkyl,
-C(0)(Ci-C6)alkyl, -COOH, -COO(Ci-C6)alkyl, -S-(C]-C6)alkyl, -S-aryl, (C]-C6)alkoxy, -
25 S0 2 NH 2 , phenyl, phenyl(C \ -C6)alkyl, -0-(C 1 -C 6 )alkyl-OH, -0-(C 1 -C6)alkyl-0-(C 1 -C 6 )alky 1, -O-
(C 2 -C 6 )alky 1-N-((C 1 -C6)alky l) n , -N-(C ] -C 6 )alky l«OH, -N-(C 1 -C 6 )alkyl-0-(C 1 ~C6)alky 1, -
C(0)NH 2 , -C(0)N((Ci-C 6 )alkyl) n , ,-S(0) n (Ci-C 6 )alkyl, -S(0) n aryl, -S(0) n heterocyclyl, and
heterocyclyl, where the alkyl groups mentioned herein optionally have one or more unsaturated
bonds in the alkyl portion;
30 n is 0, 1 or 2;
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provided that
1) when Q is H; Y is O; R 1 and R 2 are each hydrogen, halogen, alkyl, alkoxy, alkylthio,
carboxyalkyl or optionally substituted phenyl; and X 1 is hydrogen or alkyl; then R 3 is not alkyl,
alkenyl, alkoxy, cycloalkyl or optionally substituted phenyl;
2) when Q is H; Y is O; R 1 and R 2 are each hydrogen, halogen, alkyl, alkoxy, alkylthio,
carboxyalkyl or optionally substituted phenyl; then X 1 and R 3 are not taken together to form
(Z) n
or
3) when W is CI, Br or I; Q is hydrogen; Y is O; X' is H; then R 3 is not
optionally substituted by 1 to 3 substituents independently selected from the group consisting of
10 amino, mono- or bi-(C]-C6)alkylamino, hydroxy, chloro, fluoro, bromo, iodo, (C]-C6)alkyl, (C\-
C^alkoxy and -SO2NH2;
4) when W is CI, Br or I; Q is H; R l is 7-C1; R 2 is H; and X 1 is alkyl; then R 3 is not alkyl, alkoxy or
cycloalkyl;
5) when W is CI, Br or I; Q is H; R 1 is 7-C1; R 2 is H; and X 1 is H; then R 3 is not alkyl or
1 5 eye loalkylam ino;
6) when W is CI, Br, I or NO2; Q is H; Y is O; X 1 is H; Rl is OH; R 2 is NO2, amino, alkyl, alkoxy,
hydroxy lower alkyl or dialkylamino; then R 3 is not H or alkyl;
7) when W is CI, Br or I; Q is H; Y is O; R* is CF 3 , CH2F, NC>2, alkyl or alkoxy; R 2 is H; X* is H;
then R 3 is not naphthyl or phenyl optionally substituted with halo, CF3, alkyl or alkoxy;
20 8) when W is CI, Br or I; Q is H; R 1 is alkyl; R 2 is H; X 1 is H or alkyl; then R 3 is not alkyl or
alkoxy;
9) when W is CI; Q is H; Y is S; R 1 and R 2 are each H; X 1 is H; then R 3 is not ethyl; and
1 0) when W is CI; Q is H; Y is O; R 1 and R 2 are each H; X 1 is H; then R 3 is not n-butyl.
A preferred group of compounds of formula (I), designated Group A, are those compounds
25 wherein the alkyl, alkenyl and alkynyl moieties, and the alkyl portion of a moiety is an optionally
substituted straight or branched chain having one to eight carbon atoms;
17
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the aryl moiety and the aryl portion of a moiety is an optionally substituted phenyl, or
naphthyl;
the heterocyclyl moiety and the heterocyclyl portion of a moiety are selected from the group
consisting of an optionally substituted piperidinyl, pyridyl, pyrazinyl, pyrimidinyl, thienyl,
pyrrolidinyl, piperazinyl, thiomorpholinyl, morpholinyl, 2,3,4,5-tetrahydrofuranyI, 1 ,3-dioxanyl, 1,4
dioxanyl, furanyl, and 1,2,4-triazolyl, tetrazolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl,
oxadiazolyl, thiadiazolyl, benzimidazolyl, 1,3-dioxolanyI, 2-imidazolinyl, imidazolidinyl, 2-
pyrazolinyl, pyrazolidinyl, isothiazolyl, 1,2,3-triazolyl, 2H-pyranyl, 4H-pyranyl, 1,4-dithianyl, 1,3,5
triazinyl, 1,3,5-trithianyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, purinyl, 4H-quinolizinyl,
cinnolinyl, phthalazinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, 1,8-naphthpyridinyl,
pteridinyl, quinuclidinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, pyrrolyl,
isoxazolyl, pyridazinyl, indazolyl, benzoxazolyl, benzofuranyl, benzothiazolyl, indolizinyl,
imidazopyridinyl and benzothienyl.
A preferred group of compounds of Group A, designated Group B, are those compounds
wherein R 3 is an optionally substituted moiety selected from the group consisting of (C]-C8)alkyl,
phenyl, phenyl(Ci-C8)alkyl, thienyl, thienyl(Ci-C8)alkyl, piperidinyl, piperidinyl(Ci-C8)alkyl,
pyrrolidinyl, pyrrol idinyl(Ci-C8)alkyl, morpholinyl, morpholinyl(Ci-C8)alkyl, 2,3,4,5-
tetrahydrofuranyl, 2,3 5 4,5-tetrahydrofuranyl(C]-C8)alkyl, furanyl, furanyl(C]-C8)alkyl, cycloalkyl,
cycloalkyl(C]-C8)alkyl, pyridyl, pyridyl(Cj-C8)alkyl, 1,2,4-triazolyl, l,2,4-triazolyl(Ci-C8)alkyl,
A preferred group of compounds of Group B, designated Group C, are those compounds
wherein Q is H; is NO2; Y is S; R 1 is in the 7-position and is hydrogen, -CH2-S02-phenyl, -Cf-b-
CN, -CH(CH3)(CN), or -CH(CN)(CH2-phenyl); R 2 is hydrogen; X 1 is hydrogen, methyl or-
(CH 2 )2-OH;
R3 is selected from the group consisting of ethyl, benzyl, EtOH, n-PrOH, n-BuOH, n-pentanol, n-
hexanol, -(CH 2 )2-NH-(CH 2 )2-OH, -(CH 2 )2-0-(CH 2 )2-OH, -CH(CH 2 CH 3 )(CH 2 OH),
-CH(CH20H)(CH 2 -/-Pr), 2,3-di-hydroxy-propyl, 2-hydroxypropyl, -CH(CH3)(CH20H),
-C(CH 3 )2(CH 2 OH) 5 -CH2(CH3)(CH20CH 3 ), 1 ,3-dihydroxyisopropyl, -
CH(CH20H)(CH2CH2SCH3), cyclopropyl, cyclopropylmethyl, 4-hydroxycyclohexyl, 3-
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chlorophenyl, 4-chlorophenyl, 2-methylphenyl, 3-methylphenyl, 4-aminobenzyl, (4-
aminophenyl)ethyI, -(CH2)3-N(Et)2, -(CH2)2"N(Me)2, N-piperidinyl, 2,6-dimethylpiperidinyl :
'2'2
r^y -ch 2 — - ch 2 -^j/ " cH2— o < ch 2)-\^j
Another preferred group of compounds of Group B, designated Group D, are those
compounds wherein Y is O; R 1 is in the 7-position and is hydrogen, -CH2-SC>2-phenyl, -CH2-CN, -
CH(CH3)(CN) ? or -CH(CN)(CH2-phenyl); R 2 is hydrogen; X 1 is hydrogen, methyl or -(CH2)2~
OH;
R3 is selected from the group consisting of benzyl, EtOH, n-PrOH, /-BuOH, n-hexanol 5 aminoethyl,
aminopropyl, -(CH 2 )2-NH-(CH 2 )2-OH, -(CH 2 )2-0-(CH 2 )2-OH, -CH(CH 2 CH3)(CH 2 OH),
-CH(CH 2 OH)(CH 2 -/-Pr) s 2,3-di-hydroxy-propyl, 2-hydroxypropyl, -CH(CH3)(CH 2 OH), 1,3-
dihydroxyisopropyl, -CH(CH 2 OH)(CH2CH 2 SCH3), cyclobutyl, 4-hydroxycyclohexyl,
-CH(COOEt)(CH2)2-SCH 3 , -(CH 2 )2-COOEt, -(CH 2 )5-COOEt, (2-aminophenyl)methyl, 4-
aminobenzyl, (4-aminophenyl)ethyl, -C(CH3)2(phenyl), -CH2(2,4-difluorophenyI), 2-
pyridylmethyl, 3-pyridylmethyl, 4-pyridyImethyl -(CH2)2"thien-2-yl, -CH(/-Pr)(COOEt), -CH(/-
Pr)(CH 2 OH), 3-(N-methyIamino)propyl, -(CH 2 ) 3 -N(Et) 25 -(CH 2 )4-N(Et) 2? -CH(Me)(CH 2 )4-CH 3 ,
-CH(Me)(CH2) 3 -N(Et)2 ? N-piperidinyl, -(CH2)2"(4-(S02NH 2 )phenyl), 2,6-dimethylpiperidinyl,
O
P -(CH 2 ) 2 -N^> — ^^N^Ph "( CH 2)3— -(CH 2 ) 3 — N^O
N-COOEt -(CH 2 )— I
J and N
Another preferred group of compounds of Group B, designated Group E, are those
compounds wherein W is NO2; Q is hydrogen; R 1 is in the 7-position and is -CH 2 -C0 2 -t-Bu 9 allyl
or benzyl; R 2 are each hydrogen; X* is hydrogen; and R 3 is ethyl.
Another preferred group of compounds of Group B, designated Group F, are those
compounds wherein W is NO2; R 1 is in the 7-position and is hydrogen, -CH(CH3)(CN) or -
CH(CN)(CH2-phenyl); R 2 is hydrogen; and
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Y
10
15
20
N
N — R"
N
Q is taken together with X 1 and to form , where Y is O and R.3 is ethyl.
Another preferred group of compounds of Group A, designated Group G 5 are those
compounds wherein W isNC>2; Q is H; R 1 and R 2 are each hydrogen; and
r3 and X 1 are taken together with the nitrogen atom to which they are attached to form
/ \
N N-Me
\ /
or
/ \
■N N-COO-t-Bu
\ /
A preferred group of compounds of Group B, designated Group H, are those compounds
wherein W is NO2; R 1 is hydrogen or is in the 7-position and is -CH2-CN, -CH2-CONH2 and -
CH2-COO-t-Bu; R 2 is hydrogen; X 1 is hydrogen or-CH2-OCH3; R 3 is methyl, ethyl, n-BuOK
CH2CF3, morpholino, -(CH 2 )7-N(Me)2, 2-phenyl-phenyl, n-BuOH, -CH2CF3, morpholino, -
(CH 2 )4-N(Me) 2 ,
-(CH2)2-N(Me)2 5 -(CH2)3-NHMe ? benzyl or-CH2-OCH3;
Y
^ N
N — K
N
, where Y is O and RP is
or Q is hydrogen or is taken together with X^ to form
ethyl;
or R 3 and X 1 are taken together with the nitrogen atom to which they are attached to form
•N N-Z
\_
H or Me ^ w h er e Z is methyl, 4-fluorophenyl, 2-pyridyl, 2-methoxyphenyl, -CH2-
CH=CH-phenyl or 2,4-dimethoxyphenyl.
Another preferred group of compounds of formula (I), designated Group I, are those
compounds wherein W is CI or Br; Q is H; R 3 is an optionally substituted moiety selected from the
group consisting of alkyl, alkenyl, phenyl, phenylalkyl, heterocyclyl, heterocyclyl-alkyl or
aminoalkyl.
20
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A preferred group of compounds of Group I, designated Group J, are those compounds
wherein R 3 is alkyl, haloalkyl, esteralkyl, N,N-dialkylaminoalkyl, alkenyl, phenyl, phenylalkyl,
halophenyl, alkoxyphenyl, aryloxyphenyl, thienyl-alkyl, halopyridyl, heterocyclyl, heterocyclyl-
alkyl or aminoalkyl.
5 A preferred group of compounds of Group J, designated Group K, are those compounds
wherein W is CI; R 3 is ethyl, propyl, butyl, t-butyl, 2,4,6-trichlorophenyl, 2,4-dimethoxyphenyl,
-(CH 2 ) 2 -2-thienyl, allyl, 2-bromoethyl, 2-phenoxyphenyl, 2,6-dichloropyrid-4-yl, benzyl, -(CH 2 ) 2 -
COOEt, -(CH 2 )3-N(Et) 2 , -(CH 2 ) 4 -N(Et) 2 , or -(CH 2 ) 2 -N(Me) 2 .
A preferred group of compounds of Group K, designated Group L, are those compounds
10 wherein R 3 is -(CH 2 ) 2 -2-thienyl, allyl, 2-bromoethyl, 2-phenoxyphenyl, 2,6-dichloropyrid-4-yl,
benzyl, -(CH 2 ) 2 -COOEt, -(CH 2 ) 3 -N(Et) 2 , -(CH 2 ) 4 -N(Et) 2 , or -(CH 2 ) 2 -N(Me) 2 .
Another preferred group of compounds of Group J, designated Group M, are those
compounds wherein R 1 is hydroxy, nitro, or an optionally substituted moiety selected from the
group consisting of alkyl, alkoxy, arylalkyloxy and sulfonato; R 2 is halo or nitro; and R 3 is alkyl or
15 phenylalkyl.
A preferred group of compounds of Group M, designated Group N, are those compounds
wherein R 1 is hydroxy, nitro, methyl, methoxy, isopropoxy, benzyloxy, 4-fluorobenzyloxy, -O-
C(CH 3 ) 2 (C(0)NH 2 ), -0-(CH 2 ) 2 -0-(CH 2 ) 2 -OMe or~0-S0 2 -CF 3 ; R 2 is CI or nitro; and R 3 is ethyl
or benzyl.
20 A preferred group of compounds of Group N, designated Group O, are those compounds
wherein is H .
A preferred group of compounds of Group O, designated Group P, are those compounds
wherein W is CI; R 1 is in the 7-position; and R 2 is in the 4- or 5-position.
In another aspect, the present invention is directed to a compound of the formula
H
25
N — R 3
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharmaceutical ly-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein
R 3 is ethyl, propyl, t-butyl, 2,4,6-trichlorophenyl or 2,4-dimethoxyphenyl.
In another aspect the present invention is directed to a compound of the formula
21
WO 01/57008
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R 1
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein R 1
is methyl, methoxy or isopropoxy.
In another aspect, the present invention is directed to a compound of the formula (I A),
Y
(IA),
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein
10 WisN02orCN;
Y is O or S;
Rl is in the 7-position and is hydrogen, methyl, ethyl, allyl, phenyl, benzyl, -CH2-C(0)-CH3, -CH2-
C0 2 -t-Bu, -CH2-S02-aryl, -alkyl-CN, or -alkyl(CN)(CH2-aryl);
X* is hydrogen, alkyl or hydroxyalkyl;
15 R 3 is selected from the group consisting of ethyl, n-butyl, t-butyl, n-propyl, allyl, hydroxyalkyl,
aminoalkyl, -alkyl-NH-alkyl-OH, -alkyl-O-alkyl-OH, di-hydroxyalkyl, alkoxyalkyl,
(alkylthio)hydroxyalkyl, cycloalkyl, cycloalkylalkyl, hydroxycycloalkyi, (alkylthio)(alkylester)alkyl,
alkylesteralkyl, 2,4-dimethoxyphenyI, 3,5-trifluoromethylphenyl, 3-chIorophenyl, 4-chlorophenyl
2,6-dichlorophenyl, 2-methylphenyl, 3-methylphenyl, (substituted phenyl)alkyl, phenylalkyl,
20 heterocyclylalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl, optionally substituted heterocyclyl,
and optionally substituted heterocyclylalkyl.
A preferred group of compounds of formula (I A), designated Group Q, are those
compounds wherein Rl is hydrogen and X 1 is hydrogen.
Another preferred group of compounds of formula (IA), designated Group R 5 are those
25 compounds wherein W is NO2; Q is hydrogen; R 1 is in the 7-position and is hydrogen, methyl, ethyl
22
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or phenyl; R 2 are each hydrogen; X 1 is hydrogen; and R 3 is selected from the group consisting of
ethyl, n-Bu, /-Bu, n-Pr, allyl, cyclopropyl, cyclobutyl, 2 ? 4-dimethoxyphenyl, 3,5-bis-
trifluoromethylphenyl, 3-chlorophenyl, 4-chlorophenyh 2,6-dichlorophenyl, 2-methyl phenyl and 3-
methylphenyl.
Other preferred groups of compounds of the formula (I) are as follows:
• wherein W is -(CH2)2-'NH-C(O)-NH-(C(R 10 )2)a"2: 1 q or an optionally substituted heterocyclyl;
Rl and R2 are each H; Q is H; Y is O; X 1 is H; and R3 is an optionally substituted alkyl. A
preferred group of compounds of the foregoing group is where W is:
10 -(CH 2 )2-NH-C(0)-NH-Et, -CH 2 -NH-C(0)-NH-ethyl, -CH 2 -NH 2 , -NH-phenyl 5 -C(O)-
NH 2 , -CH 2 -NH-S(0) 2 -Ph, -C(0)-NH-phenyl, -CH 2 -NH-S(0) 2 -CF 3 , -CH 2 -CN, -CH 2 -NH-
CH 2 -5-methyl-furan-2-yl, -C(0)-NH-(CH 2 ) 3 -(4-methylpiperazin-l-yl) ?
-(CH 2 )2-NH-C(0)-NH-(phenyl) 5 or -(CH2) 2 -NH-C(0)-NH-(p-toluyl). A preferred group of
compounds of the immediately foregoing group is where R- 3 is ethyl.
15 • wherein W is CN; R 1 and R 2 are each H; Q is H; Y is O; X 1 is H; and R 3 is an optionally
substituted heterocyclyl-heterocyclyl, or heterocyclyl-cycloalkyl. A preferred group of the
foregoing compounds is where R 3 is 3-(4-methylpiperazino)propyl, 2-morpholinoethyl, 3-(9-
benzyl-9-azabicyclo[3.3.1]nonyl, 6-(4-methylpiperazino)-3-pyridyl 5 3-(8-benzyl-8-
azabicyclo[3 .2.1 ]octy 1, methyl-3-(8-benzyl-8-azabicyclo[3 .2. 1 ]octyl, /err-butylcarboxy late- 1 -
20 piperidinylmethyl, 4-piperidylmethyl, /er/-butylcarboxylate-l-piperazinyl-ethyl ? 2-
piperazinoethyl, 4-(4-methylpiperazino)cyclohexyl, 3-piperidinopropyl ? 6-(4-methylpiperazino)-
3-pyridyl.
• wherein Rl and W are taken together to form
23
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Y
where is independently selected from the same group of substituents as X^, A preferred group
of the foregoing group of compounds is where is H; Q is H; Y is O; X^ is H; R^ is alkyl; and
5 X 10 is ethyl, 3-pyridyl, N-(p-Br-phenyl)~NH-, 1-piperidyl or CH3-NH-.
• wherein W is H; and R* is -S-X3, -S(0)X3 or -S(0)2X3 .
• wherein W is Br, CI or p-fluorophenoxy, Rl and R2 are each H; Q is H; Y is O; X^ is H; and R^
is alkyl-chloro,
-alkyl
? ? 9
-alkyl-piperazin-f-yl, -aIkyI-(2,5-dimethyIpiperazin-l-yl), -alkyl-(3,5-dimethylpiperazin-l-yl) ; -
alkyl-(3-am inocarbony lpiperidin- 1 -y 1), -alkyl-(4-hydroxypiperidin- 1 -y 1), -alky l-(3 -
hydroxypiperidin-l-yl), -alkyl-COOEt, -alkyl-COOH, -alkyl-(4-methylpiperazin-l-yl), -alkyl-
15 (N-morphoIinoethylamino), -alkyI-(N-piperidinylethylamino), -aIkyi-(N-(N ? N-
diethylaminoethyl)-N-(methyl)amino), -alkyl-((l-ethylpyrrolidin-2-yl)-methylamino) ? -alkyl-
(N-(l-methylpiperidin-4-yl)-N-(methyl)amino) 5 -alkylamino, -alkyl-piperidin-l-yl or -alkyl-
(N,N-diethyIaminoethylamino). A preferred group of the foregoing compounds is where the
alkyl group is methylene, ethylene or propylene.
24
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• wherein R 2 is H; Q is H; Y is O; X 1 is H and R 3 is ethyl. Preferred groups of the foregoing
group of compounds is where:
• W is H or Br; and R 1 is in the 7-position of the benzothiazolyl ring and is -CsCH, -C=C-(2-
pyridinyl), -CsC-CH 2 -N(CH 3 ) 2 , -0-CH(CH 3 ) 2 , phenyl or-CH=CH 2 ;
• Rl is -CH=CH2 and W is -CH=CH2;
• RMs H and W is benzyl, p-fluorophenoxy or pyridin-4-ylmethyl;
• W is F; Rl is in the 7-position of the benzothiazolyl ring and is H or CI; and R 2 is in the 5-
position of the benzothiazolyl ring and is H or CI; or
. Rl is H and W is -CHsCH, -OC-Ph, -C=C-CH 2 -N(CH 3 ) 2 , -C=C-(4-fluorophenyl), -OC-
(p-toluyl), -(CH 2 ) 2 -Ph, -(CH 2 ) 2 -(4-fluorophenyl), -CH=CH-phenyl, -CH=CH-CH 2 -
N(CH 3 )2, -CH=CH-(4-fluorophenyl), -CH=CH-(p-toluyl), or-CH=CH-(l-imidazolyl).
• wherein W is p-fluorophenoxy, -(CH2) 3 -NHMe or -{CF^-l-piperazinyl; and R 3 is -CH 2 -
C(Me) 2 -CH2-N(CH 3 )2, -(CH 2 ) 2 -(5-im idazolyl),
15
• wherein R 1 is in the 7-position of the benzothiazolyl ring and is H or CN; R 2 is H; Y is O; Q anc
X* are each H;
W is CI, NO2, -CH2-OH, -CH2-0-C(0)-NH-Et, -S-phenyl, -O-phenyl, -S-CH 3 , -C(0)-phenyl, -
S(0)-phenyl, -S-p-nitrophenyl, -S-p-methylphenyl, -S-p-chlorophenyl, -S-/?-methoxyphenyl, -S-
m-CF 3 -phenyl, -S-o-chlorophenyl, -C(0)-CH 3 , -NH-C(0)-NH-(-CH 2 ) 2 -2-thienyl, -NH-C(O)-
NH-3-pyridyl, -S(0) 2 -/»-(carboxymethylamino)-phenyl, -N-morpholino, -NH-C(0)-NH-Et, -
25
WO 01/57008
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NH-C(0)-NH-CH2-phenyl 5 -S-/?-chlorophenyl, -S-/?-bromophenyl, -S-/W-CF3 -phenyl, or -S-p-
fluorophenyl;
(CH2)2-N-morpholino,or -CH2-piperidin-4-yl.
. wherein Q and Xl are each H; Y is O; R3 is ethyl; W is H, -OCF3, -O-Et, F, CH3, -OCH3, -
S0 2 -Me, NH 2 , -NH-C(0)-Me, -NH-CH 2 -phenyl ? -MH-S(0) 2 -2-thienyl ? -NH-S(0) 2 -(3,5-
dimethylisoxazol-4-yl), -NH-S(0) 2 -Me, -NH-S(0)2-CH 2 -phenyl, -NH-C(0)-0-CH 2 -CCl 3 , -
NH-C(0)-0-CH 2 -Ph, -NH-C(0)-0-Me orN02;
Rl is H, F or -CH 2 -S(0)2-phenyl; and
R2 is H, 4-C1, 4-methyl, 5-methyl, 5-C1, 5-F or 5-OCH 3 .
In another aspect, the present invention is directed to a method of using a compound of
formula (IB) or a pharmaceutical^ acceptable salt thereof as a replacement therapy for anti-
inflammatory glucocorticosteroid therapy in a patient undergoing anti-inflammatory
glucocorticosteroid therapy comprising the step of replacing a glucocorticosteroid with a compound
of formula (IB) or a pharmaceutically acceptable salt thereof.
Similarly, instead of a replacement therapy, a compound of the present invention can be
used in conjunction with a glucocorticoid therapy as a means of "glucocorticoid sparing" to reduce
deleterious side effects associated with glucocorticoid therapy.
In another aspect, the present invention is directed to a method of inhibiting protein kinase
activity, which comprises administering to a patient a compound of formula (IB),
26
WO 01/57008
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(IB),
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharrnaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein,
Q is H or represents a bond which is taken together with X 1 and the two nitrogen atoms to which Q
and X 1 are attached and the OY group to which the two nitrogen atoms are attached to form
Y
N
N — R
N
I,
Q
Ql is (C!-C6)alkyl;
Y is O or S;
10 W is H, CI, Br, I, N0 2 , CN, SCN, OCF3, -X q -(C(RlO) 2 )a-Y 1 q-(C(RlO) 2 ) a -Z 1 q , or an optionally
substituted group selected from the group consisting of alkyl, alkenyl, alkynyl, heterocyclyl-alkenyl,
and heterocyclyl-alkynyl;
Y 1 and X are each independently selected from the group consisting of phenyl, heterocyclyl,
NR 10 , O, S, SO, S0 2 , CF 2 , CFR, C=0, (C=O)NRl0, SONRlO SO 2 NRl0, NRl0(C=O),
15 NRIOSO,
R
I
N
10
NRl 0 SO 2 , NRl 0so 2 NRl 0, NRl 0(C=O)NRl 0,
O
R
I
N
10
R
I
N
10
R
I
N
10
O 0
and
o 0
27
WO 01/57008
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q for each occurrence is independently 0 or 1;
a for each occurrence is independently 0 or an integer from 1 to 5;
RlO f or eac h occurrence is independently selected from the group consisting of H,
optionally substituted aryl, optionally substituted heterocyclyl and an optionally substituted
alkyl group optionally substituted with one or more of the following: a C]_6 alkyl group
optionally substituted by one or more hydroxy, halo or optionally substituted amino; a C]_6
alkoxy group optionally substituted by one or more hydroxy, halo or optionally substituted
amino; hydroxy; halo; or optionally substituted amino;
Z 1 is H, optionally substituted alkyl, optionally substituted aryl or optionally substituted
heterocyclyl;
X 1 is hydrogen, alkyl, hydroxyalkyl or represents a bond which is taken together with R 3 as
described below or represents a bond which is taken together with Q as described above;
Rl and are each independently hydrogen, halogen, hydroxy, nitro, cyano, COOH, COOX 3 , SX- 3 ,
S0 2 X 3 , SOX 3 , C(0)X 3 , NHC(0)X 3 , C(0)NHX 3 , NHSO2X 3 or selected from an optionally
substituted group consisting of alkyl, alkenyl, alkynyl, alkoxy, amino, NHX 3 , NX 3 X 3 , alkylamino,
arylamino, heterocyclylamino, alkylthio, alkylsulfonato, aryl, aryloxy, arylalkyl, arylalkenyl,
arylalkynyl, arylalkyloxy, heterocyclyl, heterocyclyloxy, heterocyclyl-alkyl, heterocyclyl-alkenyi,
heterocyclyl-alkynyl, heterocyclyl-alkyloxy, heterocyclylthio, heterocyclylsulfinyl,
heterocyclylsulfonyl, cycloalkyl, -(CH 2 ) m -(CHX2)CN, -(CH 2 ) m -(CHX2)COOH, -(CH 2 )nr
(CHX2)COOX 3 , -(CH 2 ) m -(CHX2)S0 2 x3 ? »(CH 2 ) m -(CHX2)C(0)X 3 , -(CH 2 ) m -
(CHX2)C(0)NHX 3 and
-(CH 2 ) m -(CHX2)NHS0 2 X 3 ;
where m is 0 to 4;
X 2 for each occurrence is independently H or an optionally substituted moiety selected from
the group consisting of alkyl, alkenyl, alkynyl, carbonyl, S(0) p alkyl, S(0) p aryl,
S(0) p heterocycIyl, amino, alkoxy, alkylthio, arylthio, perhaloalkyl, aryl, aryloxy, arylalkyl,
arylalkyloxy, heterocyclyl and heterocyclyl-alkyl;
p is 0, 1 or 2;
X 3 for each occurrence is independently H or an optionally substituted moiety selected from
the group consisting of mono- or di-alkylamino, alkyl, alkenyl, alkynyl, aryl, arylalkyl,
heterocyclyl and heterocyclyl-alkyl;
WO 01/57008
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or when R 1 is in the 7-position of the benzothiazole ring, R 1 and W can be taken together with the
carbon atoms to which they are attached to form an optionally substituted 5- or 6-mernbered
heterocyclyl ring;
r3 i s hydrogen, or an optionally substituted moiety selected from the group consisting of carbonyl,
5 alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heterocyclyl, heterocyclyl-alkyl, heterocyclyl-
heterocyclyl, heterocyclyl-cycloalkyl, amino, alkylamino, arylamino, alkoxy, thioalkoxy and acyl;
or R 3 and are taken together with the nitrogen atom to which they are attached to form
s
where Z for each occurrence is independently selected from the group consisting of oxo, or
10 an optionally substituted moiety selected from the group consisting of -C(0)(Ci-C6)alkyl,
-C(0)ary 1, -C(0)N(C ] -C 6 )alkyl, -C(0)N-aryl, (C j -C 6 )alkyl, (C 2 -C6)alkeny 1, (C 2 -
C6)alkynyl, amino, mono- or di-(Ci-C6)alkylamino, -COO(C j-C6)alkyl, pyridyl, phenyl,
phenyl(C]-C6)alkyl and phenyl(Ci-C6)alkenyI;
where each of the optionally substituted moieties described hereinabove is optionally substituted by
15 one or more substituents each independently selected from the group consisting of oxo, amino, nitro,
mono- or bi-(Ci-C6)alkylamino, hydroxy, nitrile, chloro, fluoro, bromo, iodo, CF3, (Ci-C6)alkyl,
-C(0)(C]-C 6 )alkyl, -COOH, -COO(C 3 -C6)alkyl, -S-(C]-C 6 )alkyl, -S-aryl, (Ci-C6)alkoxy, -
S0 2 NH 2 , phenyl, phenyl(Ci-C6)alkyl, -0-(Ci-C 6 )alkyl-OH, -0-(Ci-C6)alkyl-0-(C 1 -C 6 )aIkyl, -O-
(C 2 -C 6 )alky 1-N-((C 1 -C 6 )alky l) n , -N-(C 1 -C 6 )alky 1-OH, -N-(C 1 -C 6 )alkyl-0-(C ] -C 6 )alkyl, -
20 C(0)NH 2? -C(0)N((Ci-C6)alkyl) n , ,-S(0) n (Ci-C6)alkyl, -S(0) n aryl, -S(0) n heterocyclyl ? and
heterocyclyl, where the alkyl groups mentioned herein optionally have one or more unsaturated
bonds in the alkyl portion;
n is 0, 1 or 2.
A preferred embodiment of a compound of formula (IB) is
WO 01/57008
PCT/US01/03803
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein,
Q is H or represents a bond which is taken together with X 1 and the two nitrogen atoms to which Q
and X 1 are attached and the C=Y group to which the two nitrogen atoms are attached to form
Y
£ — N^^N — R 3
N
I,
5 ;
Ql is(Ci-C6)alkyl;
Y is O or S;
W is CI, Br, I, N0 2 or CN;
where X^ is hydrogen, alkyl, hydroxyalkyl or represents a bond which is taken together with
10 r3 as described below or represents a bond which is taken together with Q as described
above;
Rl and R 2 are each independently hydrogen, halogen, hydroxy, nitro, cyano, COOH, COOX 3 ,
S0 2 X 3 , SOX 3 , C(0)X 3 , NHC(0)X 3 , C(0)NHX 3 , NHSO2X 3 or selected from an optionally
substituted group consisting of alkyl, alkenyl, alkynyl, alkoxy, amino, NHX 3 , NX 3 X 3 , alkylamino,
15 arylamino, heterocyclylamino, alkylthio, alkylsulfonato, aryl, aryloxy, arylalkyl, arylalkenyl,
arylalkynyl, arylalkyloxy, heterocyclyl, heterocyclyloxy, heterocyclyl-alkyl, heterocyclyl-alkenyl,
heterocyclyl-alkynyl, heterocyclyl-alkyloxy, heterocyclylthio, heterocyclylsulfinyl,
heterocyclylsulfonyl, cycloalkyl, -(CH 2 ) m -(CHX 2 )CN, -(CH 2 ) m -(CHX 2 )COOH, -(CH 2 ) m -
(CHX 2 )COOX 3 , -(CH 2 ) m -(CHX 2 )S0 2 X 3 , -(CH 2 ) m -(CHX2)C(0)X 3 , -(CH 2 ) m -
20 (CHX 2 )C(0)NHX 3 and
-(CH 2 ) m -(CHX2)NHS0 2 X 3 ;
where m is 0 to 4;
X 2 for each occurrence is independently H or an optionally substituted moiety selected from
the group consisting of alkyl, alkenyl, alkynyl, carbonyl, S(0) p alkyl, S(0) p aryl,
25 S(0) p heterocyclyl, amino, alkoxy, alkylthio, arylthio, perhaloalkyl, aryl, aryloxy, arylalkyl,
arylalkyloxy, heterocyclyl and heterocyclyl-alkyl;
p is 0, 1 or 2;
30
WO 01/57008 PCT/US01/03803
X 3 for each occurrence is independently H or an optionally substituted moiety selected from
the group consisting of mono- or di-alkylamino, alkyl, alkenyl, alkynyl, aryl, arylalkyl,
heterocyclyl and heterocyclyl-alkyl;
R3 is hydrogen, or an optionally substituted moiety selected from the group consisting of carbonyl,
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heterocyclyl, heterocyclyl-alkyl, amino,
alkylamino, arylamino, alkoxy, thioalkoxy and acyl;
or R 3 and X 1 are taken together with the nitrogen atom to which they are attached to form
N or
where Z for each occurrence is independently .selected from the group consisting of oxo, or
10 an optionally substituted moiety selected from the group consisting of-C(0)(Ci-C6)alkyl,
-C(0)aryl, -C(0)N(C i-C6)alkyl, ~C(0)N~aryl, (C i -C6)alkyl, (C 2 -C6)alkenyl, (C 2 -
C6)alkynyl, amino, mono- or di-(C)-C6)alkylamino, -COO(C]-C6)alkyl, pyridyl, phenyl,
phenyl(Ci-C6)alkyl and phenyl(Ci-C6)alkenyl;
where each of the optionally substituted moieties described hereinabove is optionally substituted by
15 one or more substituents each independently selected from the group consisting of oxo, amino, nitro,
mono- or bi-(C j-C6)aIkyIamino, hydroxy, nitrite, chloro, fluoro, bromo, iodo, CF3, (C]-C6)alkyl,
-C(0)(Ci-C 6 )alkyl, -COOH, -COO(Ci-C 6 )alkyl, -S-(C^C 6 )aIkyl, -S-aryl, (C]-C 6 )alkoxy, -
S0 2 NH 2 , phenyl, phenyI(Ci-C 6 )alkyl, -0-(C i-C 6 )alkyl-OH, -0-(Ci-C 6 )alkyl-0-(Ci-C 6 )alkyl, -O-
(C 2 -C 6 )alkyl-N-((C ] -C 6 )alkyl) n , -N-(C ] -C 6 )alkyl-OH, -N-(C j ~C 6 )alkyl-0-(C 1 -C 6 )alkyl, -
20 C(0)NH 2 , -C(0)N((Ci-C 6 )alkyl) n5 ,-S(0) n (Ci-C 6 )alkyl -S(0) n aryl, -S(0) n heterocyclyl, and
heterocyclyl, where the alkyl groups mentioned herein optionally have one or more unsaturated
bonds in the alkyl portion;
n is 0, 1 or 2.
A preferred method of the immediately foregoing method is where said protein kinase is a
25 tyrosine kinase.
A preferred method of the immediately foregoing method is where said tyrosine kinase is a
receptor tyrosine kinase or a non-receptor tyrosine kinase.
A preferred method of the immediately foregoing method is where tyrosine kinase is KDR
or Lck.
31
WO 01/57008
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Another preferred method of inhibiting a tyrosine kinase with a compound of formula (IB) is
where the tyrosine kinase affects angiogenesis.
A preferred method of the immediately foregoing method is where the inhibition of said
tyrosine kinase results in an anti-angiogenic effect.
5 In another aspect, the present invention is directed to a method of treating a condition,
disorder or disease, which comprises administering to a patient a compound of formula (IB), as
defined hereinabove, where said condition, disorder or disease is selected from the group consisting
of hyperproliferative disorders, an ulcer, Lyme disease, sepsis, von Hippel Lindau disease,
pemphigoid, psoriasis, Paget's disease, polycystic kidney disease, fibrosis, sarcoidosis, cirrhosis,
10 thyroiditis, hyperviscosity syndrome, Osier- Weber-Rendu disease, chronic occlusive pulmonary
disease, ovarian hyperstimulation syndrome, preeclampsia, menometrorrhagia, endometriosis,
chronic inflammation, systemic lupus, glomerulonephritis, synovitis, inflammatory bowel disease,
Crohn's disease, glomerulonephritis, rheumatoid arthritis, osteoarthritis, multiple sclerosis, graft
rejection, sickle cell anaemia, an ocular condition, a cardiovascular condition, atherosclerosis,
15 restenosis, ischemia/reperfusion injury, vascular occlusion, carotid obstructive disease, cancer,
Crow-Fukase (POEMS) syndrome, a diabetic condition, anemia, ischemia, infarct, transplant
rejection, a wound, gangrene, necrosis, asthma or edema following burns, trauma, radiation, stroke,
hypoxia or ischemia, and infection by Herpes simplex, Herpes Zoster, human immunodeficiency
virus, parapoxvirus, protozoa or toxoplasmosis.
20 A preferred method of the immediately foregoing method is where:
the ocular condition is ocular or macular edema, ocular neovascular disease, scleritis, radial
keratotomy, uveitis, vitritis, myopia, optic pits, chronic retinal detachment, post-laser
treatment complications, conjunctivitis, Stargardt's disease, Eales disease, retinopathy or
macular degeneration;
25 the cancer is a solid tumor, a sarcoma, fibrosarcoma, osteoma, melanoma, retinoblastoma, a
rhabdomyosarcoma, glioblastoma, neuroblastoma, teratocarcinoma, an hematopoietic
malignancy, malignant ascites, Kaposi's sarcoma, Hodgkin's disease, lymphoma, myeloma
or leukemia; and
the diabetic condition is insulin-dependent diabetes mellitus glaucoma, diabetic retinopathy
30 or microangiopathy.
In another aspect, the present invention is directed to a method of decreasing fertility in a
patient, which comprises administering to a patient an effective amount of a compound of formula
(IB), as defined hereinabove.
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In another aspect, the present invention is directed to a method of promoting angiogenesis or
vasculogenesis, which comprises administering to a patient a compound of formula (IB), as defined
hereinabove.
A preferred method of the immediately foregoing method is where the compound of formula
5 (IB) is administered in combination with a pro-angiogenic growth factor,
In another aspect, the present invention is directed to a method of treating a patient having a
condition which is mediated by protein kinase activity, said method comprising the step of
administering to the patient a therapeutically effective amount of a compound of formula (IB), as
defined hereinabove.
10 A preferred method of the immediately foregoing method is where the protein kinase
activity is involved in T cell activation, B cell activation, mast cell degranulation, monocyte
activation, the potentiation of an inflammatory response or a combination thereof.
In another aspect, the present invention is directed to a pharmaceutical composition
comprising a compound of formula (I), as defined hereinabove, and a pharmaceutically acceptable
15 diluent or carrier.
In another aspect, the present invention is directed to a pharmaceutical composition
comprising an effective amount of a compound of formula (IB) for inhibiting a protein kinase and a
pharmaceutically acceptable carrier or diluent.
The compounds of this invention are useful as inhibitors of serine/threonine and tyrosine
20 kinases. In particular, compounds of this invention are useful as inhibitors of tyrosine kinases that
are important in hyperproliferative diseases, especially in cancer and in the process of angiogenesis.
For example, certain of these compounds are inhibitors of such receptor kinases as KDR, Fit- 1 ,
VEGFR-3, FGFR, PDGFR, c-Met, Tie-2, Tie-1 or IGF-l-R. Since certain of these compounds are
anti-angiogenic, they are important substances for inhibiting the progression of disease states, such
25 as cancer, arthritis and ocular neovascularization, where angiogenesis is an important component.
Since certain of these agents block the responses to VEGFs, and because VEGF is strongly
upregulated under conditions of hypoxia, these compounds are useful in controlling the vascular
leakage and neovascular events following ischemia and tissue damage. Certain compounds of the
invention are effective as inhibitors of such serine/threonine kinases as PKCs, erk, MAP kinases,
30 cdks, Plk-1 or Raf-L These compounds are useful in the treatment of cancer, and hyperproliferative
disorders. In addition, certain compounds are effective inhibitors of non-receptor kinases such as
those of the Src (for example, Ick, blk and lyn), Tec, Csk, Jak, Map, Nik and Syk families. These
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compounds are useful in the treatment of cancer, hyperproliferative disorders and immunologic
diseases.
The compounds of this invention, when administered to individuals in need of such
compounds, inhibit vascular hyperpermeability and the formation of edema in these individuals.
5 These compounds act, it is believed, by inhibiting the activity of KDR tyrosine kinase which is
involved in the process of vascular hyperpermeability and edema formation. The KDR tyrosine
kinase may also be referred to as FLK-1 tyrosine kinase, NYK tyrosine kinase or VEGFR-2 tyrosine
kinase. KDR tyrosine kinase is activated when vascular endothelial cell growth factor (VEGF) or
another activating ligand (such as VEGF-C, VEGF-D, VEGF-E or HIV Tat protein) binds to a KDR
10 tyrosine kinase receptor which lies on the surface of vascular endothelial cells. Following such KDR
tyrosine kinase activation, hyperpermeability of the blood vessels occurs and fluid moves from the
blood stream past the blood vessel walls into the interstitial spaces, thereby forming an area of
edema. Diapedesis also often accompanies this response. Similarly, excessive vascular
hyperpermeability can disrupt normal molecular exchange across the endothelium in critical tissues
15 and organs (e.g., lung and kidney), thereby causing macromolecular extravasation and deposition.
Following this acute response to KDR stimulation which is believed to facilitate the subsequent
angiogenic process, prolonged KDR tyrosine kinase stimulation results in the proliferation and
chemotaxis of vascular endothelial cells and formation of new vessels. By inhibiting KDR tyrosine
kinase activity, either by blocking the production of the activating ligand, by blocking the activating
20 ligand binding to the KDR tyrosine kinase receptor, by preventing receptor dimerization and
transphosphorylation, by inhibiting the enzyme activity of the KDR tyrosine kinase (inhibiting the
phosphorylation function of the enzyme) or by some other mechanism that interrupts its downstream
signaling (D. Mukhopedhyay et al., Cancer Res. 58:1278-1284 (1998) and references therein),
hyperpermeability, as well as associated extravasation, subsequent edema formation and matrix
25 deposition, and angiogenic responses, may be inhibited and minimized.
One group of preferred compounds of this invention have the property of inhibiting KDR
tyrosine kinase activity without significantly inhibiting Fit- 1 tyrosine kinase activity (Fit- 1 tyrosine
kinase is also referred to as VEGFR-1 tyrosine kinase). Both KDR tyrosine kinase and Flt-1
tyrosine kinase are activated by VEGF binding to KDR tyrosine kinase receptors and to Flt-1
30 tyrosine kinase receptors, respectively. Since Flt-1 tyrosine kinase activity may mediate important
events in endothelial maintenance and vascular function, an inhibition of this enzyme activity may
lead to toxic or adverse effects. At the very least, such inhibition is unnecessary for blocking the
angiogenic responses, induction of vascular hyperpermeability and the formation of edema, so it is
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wasteful and of no value to the individual. Certain preferred compounds of this invention are unique
because they inhibit the activity of one VEGF-receptor tyrosine kinase (KDR) that is activated by
activating ligands but do not inhibit other receptor tyrosine kinases, such as Fit- 1 , that are also
activated by certain activating ligands. The preferred compounds of this invention are, therefore,
selective in their tyrosine kinase inhibitory activity.
The compounds of the present invention are also useful in the treatment of ulcers - bacterial, fungal,
Mooren ulcers and ulcerative colitis.
Certain compounds of this invention are Tie-2 and/or Tie-1 kinase inhibitors which may be
anti-angiogenic (especially in combination with inhibition of VEGFR), or pro-angiogenic, when
employed in the presence of, or in conjunction with, a VEGF-related stimulus, in this manner such
inhibitors can be used in the promotion of therapeutic angiogenesis to treat, for example, ischemia,
infarct or occlusion, or to promote wound healing.
The present invention provides a method of inhibiting the kinase activity of tyrosine kinases
and serine/threonine kinases comprising the administration of a compound represented by formula I
to said kinase in sufficient concentration to inhibit the enzyme activity of said kinase.
The present invention further includes pharmaceutical compositions of the compounds
described herein comprising a pharmaceutical^ effective amount of the compounds and a
pharmaceutically acceptable carrier or excipient. These pharmaceutical compositions can be
administered to individuals to slow or halt the process of angiogenesis in angiogenesis-aided
diseases, or to treat edema, effusions, exudates or ascites and other conditions associated with
vascular hyperpermeability. Certain pharmaceutical compositions can be administered to individuals
to treat cancer and hyperproliferative disorders by inhibiting serine/threonine kinases such as cdk,
Plk-1, erk, etc.
In the treatment of malignant disorders, combinations with antiproliferative or cytotoxic
chemotherapies or radiation are anticipated.
DETAILED DESCRIPTION OF THE INVENTION
Certain of the compounds of this invention have antiangiogenic properties. These
antiangiogenic properties are due at least in part to the inhibition of protein tyrosine kinases essential
for angiogenic processes. For this reason, these compounds can be used as active agents against
such disease states as arthritis, atherosclerosis, restenosis, psoriasis, hemangiomas,
hemangioendothelioma, myocardial angiogenesis, coronary and cerebral collaterals, ischemic limb
angiogenesis, ischemia/reperfusion injury, wound healing, peptic ulcer Helicobacter related diseases,
virally-induced angiogenic disorders, fractures, Crow-Fukase syndrome (POEMS), preeclampsia,
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menometrorrhagia, cat scratch fever, rubeosis, neovascular glaucoma and retinopathies such as those
associated with diabetic retinopathy, retinopathy of prematurity, or age-related macular
degeneration. In addition, some of these compounds can be used as active agents against solid
tumors, malignant ascites, von Hippel Lindau disease, hematopoietic cancers and hyperproliferative
5 disorders such as thyroid hyperplasia (especially Grave's disease), and cysts (such as follicular cysts
or hypervascularity of ovarian stroma characteristic of polycystic ovarian syndrome (Stein-
Leventhal syndrome)) and polycystic kidney disease since such diseases require a proliferation of
blood vessel cells for growth and/or metastasis.
Further, some of these compounds can be used as active agents against burns, chronic lung
10 disease, stroke, polyps, asthma, anaphylaxis, chronic and allergic inflammation, delayed-type
hypersensitivity, ovarian hyperstimulation syndrome, brain tumor-associated cerebral edema, or
cerebral or pulmonary edema induced by high altitude trauma or hypoxia, ocular and macular
edema, ascites, glomerulonephritis and other diseases where vascular hyperpermeability, effusions,
exudates, protein extravasation, or edema is a manifestation of the disease. The compounds will also
15 be useful in treating disorders in which protein extravasation leads to the deposition of fibrin and
extracellular matrix, promoting stromal proliferation (e.g. keloid, fibrosis, cirrhosis and carpal tunnel
syndrome). Increased VEGF production potentiates inflammatory processes such as monocyte
recruitment and activation. The compounds of this invention will also be useful in treating
inflammatory disorders such as inflammatory bowel disease (IBD) and Crohn's disease.
20 The compounds of the present invention are in particular applicable to the treatment of an
inflammatory rheumatoid or rheumatic disease, especially of manifestations at the locomotor
apparatus, such as various inflammatory rheumatoid diseases, especially chronic polyarthritis (=
rheumatoid arthritis (very preferred)), including juvenile arthritis or psoriasis arthropathy;
paraneoplastic syndrome or tumor-induced inflammatory diseases, turbid effusions, collagenosis,
25 such as systemic Lupus erythematosus, poly-myositis, dermato-myositis, systemic sclerodermia or
mixed collagenosis; postinfectious arthritis (where no living pathogenic organism can be found at or
in the affected part of the body), or seronegative spondylarthritis, such as spondylitis ankylosans;
vasculitis; sarcoidosis; peritoneal sclerosis (especially in dialysis patients); arthrosis; or further any
combinations thereof.
30 From the foregoing it will be understood that the present invention is to be further
understood as embracing the treatment, e.g. therapy of any disease or condition as set forth above,
for example rheumatoid arthritis, arthroses, dermatomyositis etc., for example, for the alleviation or
control of inflammatory processes or events and the sequelae associated therewith or consequential
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thereto, e.g. for the treatment of rheumatoid arthritis, e.g. to alleviate or control joint inflammation or
effusion.
In a further aspect it has been found in accordance with the present invention that systemic
administration of a compound of the present invention, or a salt thereof, is useful as replacement
5 therapy for anti-inflammatory glucocorticosteroid, e.g. cortizone or the like, therapy or as
glucocorticoid sparing therapy. For example for use in any means of treatment as hereinbefore set
forth.
On the basis of their efficacy as inhibitors of VEGF-receptor tyrosine kinase activity, the
compounds of the present invention primarily inhibit the growth of blood vessels and are thus, for
10 example, effective against a number of diseases associated with deregulated angiogenesis, especially
diseases caused by ocular neovascularisation, especially retinopathies, such as diabetic retinopathy
or age-related macula degeneration, psoriasis, haemangioblastoma, such as haemangioma, mesangial
cell proliferation disorders, such as chronic or acute renal diseases, e.g. diabetic nephropathy,
malignant nephrosclerosis, thrombic microangiopathy syndromes or transplant rejection, or
15 especially inflammatory renal disease, such as glomerulonephritis, especially mesangioproliferative
glomerulonephritis, haemolytic-uraemic syndrome, diabetic nephropathy, hypertensive
nephrosclerosis, atheroma, arterial restenosis, autoimmune diseases, acute inflammation, fibrotic
disorders (e.g. hepatic cirrhosis), diabetes, neurodegenerative disorders and especially neoplastic
diseases (solid tumors, but also leukemias and other hematopoietic malignancies, or "liquid
20 tumours", those expressing c-kit, KDR or flt-1), such as especially breast cancer, cancer of the
colon, lung cancer (especially small-cell lung cancer), cancer of the prostate or Kaposi's' sarcoma. A
compound of formula I (or an N-oxide thereof) inhibits the growth of tumours and is especially
suited to preventing the metastatic spread of tumours and the growth of mircometastases.
VEGFs are unique in that they are the only angiogenic growth factors known to contribute to
25 vascular hyperpermeability and the formation of edema. Indeed, vascular hyperpermeability and
edema that is associated with the expression or administration of many other growth factors appears
to be mediated via VEGF production. Inflammatory cytokines stimulate VEGF production. Hypoxia
results in a marked upregulation of VEGF in numerous tissues, hence situations involving infarct,
occlusion, ischemia, anemia, or circulatory impairment typically invoke VEGF/VPF mediated
30 responses. Vascular hyperpermeability, associated edema, altered transendothelial exchange and
macromolecular extravasation, which is often accompanied by diapedesis, can result in excessive
matrix deposition, aberrant stromal proliferation, fibrosis, etc. Hence, VEGF-mediated
hyperpermeability can significantly contribute to disorders with these etiologic features.
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Because blastocyst implantation, placental development and embryogenesis are
angiogenesis dependent, certain compounds of the invention are useful as contraceptive agents and
antifertility agents.
It is envisaged that the disorders listed above are mediated to a significant extent by protein
5 tyrosine kinase activity involving the KDR/VEGFR-2 and/or the Flt-l/VEGFR-1 and/or Flt-
4/VEGFR-3 and/or Tie-2 and/or Tie-1 tyrosine kinases. By inhibiting the activity of these tyrosine
kinases, the progression of the listed disorders is inhibited because the angiogenic or vascular
hyperpermeability component of the disease state is severely curtailed. The action of certain
compounds of this invention, by their selectivity for specific tyrosine kinases, result in a
10 minimization of side effects that would occur if less selective tyrosine kinase inhibitors were used.
Certain compounds of the invention are also effective inhibitors of FGFR, PDGFR, c-Met and IGF-
1-R kinases. These receptor kinases can directly or indirectly potentiate angiogenic and
hyperproliferative responses in various disorders, hence their inhibition can impede disease
progression.
15 Progression through the eukaryotic cell cycle is controlled by a family of kinases called
cyclin dependent kinases (CDKs) (Myerson et al, EMBO Journal 11:2909-2917 (1992)). The
regulation of CDK activation is complex, but requires the association of the CDK with a member of
the cyclin family of regulatory subunits (Draetta, Trends in Cell Biology, 3:287-289 (1993)); Murray
and Kirschner, Nature, 339:275-280 (1989); Solomon et al, Molecular Biology of the Cell, 3:13-27
20 (1992)). A further level of regulation occurs through both activating and inactivating
phosphorylations of the CDK subunit (Draetta, Trends in Cell Biology, 3:287-289 (1993)); Murray
and Kirschner, Nature, 339:275-280 (1989); Solomon et al, Molecular Biology of the Cell, 3:13-27
(1992); Ducommun et ah, EMBO Journal, 10:3311-3319 (1991); Gautier et al, Nature 339:626-629
(1989); Gould and Nurse, Nature, 342:39-45 (1989); Krek and Nigg, EMBO Journal 10:3331-3341
25 (1991); Solomon et al, Cell, 63:1013-1024 (1990)). The coordinate activation and inactivation of
different cyclin/CDK complexes is necessary for normal progression through the cell cycle (Pines,
Trends in Biochemical Sciences, 18:195-197 (1993); Sherr, Cell, 73:1059-1065 (1993)). Both the
critical Gl-S and G 2-M transitions are controlled by the activation of different cyclin/CDK
activities. In Gl, both cyclin D/CDK4 and cyclin E/CDK2 are thought to mediate the onset of S-
30 phase (Matsushima et al, Molecular & Cellular Biology, 14:2066-2076 (1994); Ohtsubo and
Roberts, Science, 259:1908-1912 (1993); Quelle et al, Genes & Development, 7:1559-1571 (1993);
Resnitzky et al, Molecular & Cellular Biology, 14:1669-1679 (1994)). Progression through S-
phase requires the activity of cyclin A/CDK2 (Girard et al, Cell, 67:1169-1179 (1991); Pagano et
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aL, EMBO Journal, 11:961-971 (1992); Rosenblatt et aL, Proceedings of the National Academy of
Science USA, 89:2824-2828 (1992); Walker and Mailer, Nature, 354:314-317 (1991); Zindy et aL,
Biochemical <£ Biophysical Research Communications, 182:1144-1154 (1992)) whereas the
activation of cyclin A/cdc2 (CDK1) and cyclin B/cdc2 are required for the onset of metaphase
5 (Draetta, Trends in Cell Biology, 3:287-289 (1993)); Murray and Kirschner, Nature, 339:275-280
(1989); Solomon et ah, Molecular Biology of the Cell, 3:13-27 (1992); Girard et aL, Cell, 67:1 169-
1 179 (1991); Pagano et aL, EMBO Journal, 11:961-971 (1992); Rosenblatts aL, Proceedings of the
National Academy of Science USA, 89:2824-2828 (1992); Walker and MzlUqt, Nature, 354:314-317
(1991); Zindy et aL, Biochemical & Biophysical Research Communications, 182:1 144-1 154 (1992)).
10 It is not surprising, therefore, that the loss of control of CDK regulation is a frequent event in
hyperproliferative diseases and cancer. (Pines, Current Opinion in Cell Biology, 4:144-148 (1992);
Lees, Current Opinion in Cell Biology, 7:773-780 (1995); Hunter and Pines, Cell, 79:573-582
(1994)). The selective inhibition of CDKs is therefore an object of the present invention.
The method of the present invention is useful in the treatment of protein kinase-mediated
15 conditions, such as any of the conditions described above. In one embodiment, the protein kinase-
mediated condition is characterized by undesired angiogenesis, edema, or stromal deposition. For
example, the condition can be one or more ulcers, such as ulcers caused by bacterial or fungal
infections, Mooren ulcers and ulcerative colitis. The condition can also be due to a microbial
infection, such as Lyme disease, sepsis, septic shock or infections by Herpes simplex, Herpes Zoster,
20 human immunodeficiency virus, protozoa, toxoplasmosis or parapoxvirus; an angiogenic disorders,
such as von Hippel Lindau disease, polycystic kidney disease, pemphigoid, Paget's disease and
psoriasis; a reproductive condition, such as endometriosis, ovarian hyperstimulation syndrome,
preeclampsia or menometrorrhagia; a fibrotic and edemic condition, such as sarcoidosis, fibrosis,
cirrhosis, thyroiditis, hyperviscosity syndrome systemic, Osier- Weber- Rendu disease, chronic
25 occlusive pulmonary disease, asthma, and edema following burns, trauma, radiation, stroke, hypoxia
or ischemia; or an inflammatory/immunologic condition, such as systemic lupus, chronic
inflammation, glomerulonephritis, synovitis, inflammatory bowel disease, Crohn's disease,
rheumatoid arthritis, osteoarthritis, multiple sclerosis and graft rejection. Suitable protein kinase-
mediated conditions also include sickle cell anaemia, osteoporosis, osteopetrosis, tumor-induced
30 hypercalcemia and bone metastases. Additional protein kinase-mediated conditions which can be
treated by the method of the present invention include ocular conditions such as ocular and macular
edema, ocular neovascular disease, scleritis, radial keratotomy, uveitis, vitritis, myopia, optic pits,
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chronic retinal detachment, post-laser complications, conjunctivitis, Stargardfs disease and Eales
disease, in addition to retinopathy and macular degeneration.
The compounds of the present invention are also useful in the treatment of cardiovascular
conditions such as atherosclerosis, restenosis, vascular occlusion and carotid obstructive disease.
5 The compounds of the present invention are additionally useful in the treatment of one or
more diseases afflicting mammals which are characterized by cellular proliferation in the areas of
blood vessel proliferative disorders, vascular malformation, lymphoproliferative disorders
lymphangiogenesis (especially Tie-2 & FIt-4/VEGFR-3 inhibitors), fibrotic disorders, mesangial cell
proliferative disorders and metabolic diseases. Blood vessel proliferative disorders include
10 inappropriate ocular neovascularization, arthritis and restenosis. Fibrotic disorders include hepatic
cirrhosis and atherosclerosis. Mesangial cell proliferative disorders include glomerulonephritis,
diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, organ
transplant rejection and glomerulopathies. Metabolic disorders include psoriasis, diabetes mellitus,
chronic wound healing, inflammation, neurodegenerative diseases, macular degeneration, and
15 diabetic retinopathy.
Inhibitors of kinases involved in mediating or maintaining these disease states represent
novel therapies for these disorders. Examples of such kinases include, but are not limited to: (1)
inhibition of c-Src (Brickell, Critical Reviews in Oncogenesis, 3:401-406 (1992); Courtneidge,
Seminars in Cancer Biology, 5:236-246 (1994), raf (Powis, Pharmacology & Therapeutics, 62:57-95
20 (1994)) and the cycl in-dependent kinases (CDKs) 1, 2 and 4 in cancer (Pines, Current Opinion in
Cell Biology, 4:144-148 (1992); Lees, Current Opinion in Cell Biology, 7:773-780 (1995); Hunter
and Pines, Cell, 79:573-582 (1994)), (2) inhibition of CDK2 or PDGF-R kinase in restenosis
(Buchdunger et al., Proceedings of the National Academy of Science USA, 92:2258-2262 (1995)),
(3) inhibition of CDK5 and GSK3 kinases in Alzheimers (Hosoi et al., Journal of Biochemistry
25 (Tokyo), 117:741-749 (1995); Aplin et al, Journal of Neurochemistry, 67:699-707 (1996), (4)
inhibition of c-Src kinase in osteoporosis (Tanakae/ ah, Nature, 383:528-531 (1996), (5) inhibition
of GSK-3 kinase in type-2 diabetes (Borthwick et al, Biochemical & Biophysical Research
Communications, 210:738-745 (1995), (6) inhibition of the p38 kinase in inflammation (Badger et
ah, The Journal of Pharmacology and Experimental Therapeutics, 279:1453-1461 (1996)), (7)
30 inhibition of VEGF-R 1-3 and Tie-1 and -2 kinases in diseases which involve angiogenesis
(Shawver et al, Drug Discovery Today, 2:50-63 (1997)), (8) inhibition of UL97 kinase in viral
infections (He et al, Journal of Virology, 71:405-411 (1997)), (9) inhibition of CSF-1R kinase in
bone and hematopoetic diseases (Myers et al, Bioorganic & Medicinal Chemistry Letters, 7:421-
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424 (1997), and (10) inhibition of Lck kinase in autoimmune diseases and transplant rejection
(Myers et ah, Bioorganic & Medicinal Chemistry Letters, 7:417-420 (1997)).
It is additionally possible that inhibitors of certain kinases may have utility in the treatment
of diseases when the kinase is not misregulated, but it nonetheless essential for maintenance of the
5 disease state. In this case, inhibition of the kinase activity would act either as a cure or palliative for
these diseases. For example, many viruses, such as human papilloma virus, disrupt the cell cycle
and drive cells into the S-phase of the cell cycle (Vousden, FASEB Journal, 7:8720879 (1993)).
Preventing cells from entering DNA synthesis after viral infection by inhibition of essential S-phase
initiating activities such as CDK2, may disrupt the virus life cycle by preventing virus replication.
10 This same principle may be used to protect normal cells of the body from toxicity of cycle-specific
chemotherapeutic agents (Stone et al, Cancer Research, 56:3199-3202 (1996); Kohn et ah, Journal
of Cellular Biochemistiy, 54:44-452 (1994)). Inhibition of CDKs 2 or 4 will prevent progression
into the cycle in normal cells and limit the toxicity of cytotoxics which act in S-phase, G2 or mitosis.
Furthermore, CDK2/cycIin E activity has also been shown to regulate NF-kB. Inhibition of CDK2
15 activity stimulates NF-kB-dependent gene expression, an event mediated through interactions with
the p300 coactivator (Perkins et ah, Science, 275:523-527 (1997)). NF-kB regulates genes involved
in inflammatory responses (such as hematopoetic growth factors, chemokines and leukocyte
adhesion molecules) (Baeuerle and Henkel, Annual Review of Immunology, 12:141-1 79 (1994)) and
may be involved in the suppression of apoptotic signals within the cell (Beg and Baltimore^c/ewce,
20 274:782-784 (1996); Wang et at, Science, 274:784-787 (1996); Van Antwerp et aL, Scie?ice,
274:787-789 (1996)). Thus, inhibition of CDK2 may suppress apoptosis induced by cytotoxic drugs
via a mechanism which involves NF-kB. This therefore suggests that inhibition of CDK2 activity
may also have utility in other cases where regulation of NF-kB plays a role in etiology of disease. A
further example may be take from fungal infections: Aspergillosis is a common infection in
25 immune-compromised patients (Armstrong, Clinical Infectious Diseases, 16:1-7 (1993)). Inhibition
of the Aspergillus kinases Cdc2/CDC28 or Nim A (Osmani et al, EMBO Journal, 10:2669-2679
(1991); Osmani et ah, Cell, 67:283-291 (1991)) may cause arrest or death in the fungi, improving
the therapeutic outcome for patients with these infections.
In one embodiment, the present invention provides compounds of formulas (I), (I A) and
30 (IB) as described above.
Compounds of formulas (I) ? (IA) and (IB) may exist as salts with pharmaceutically
acceptable acids. The present invention includes such salts. Examples of such salts include but are
not limited to hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates,
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acetates, citrates, fumarates, tartrates [eg (+)-tartrates, (-)-tartrates or mixtures thereof including
racemic mixtures], succinates, benzoates and salts with amino acids such as glutamic acid. These
salts may be prepared by methods known to those skilled in the art.
Certain compounds of formulas (I), (IA) and (IB) which have acidic substituents may exist
as salts with pharmaceutically acceptable bases. The present invention includes such salts. Example
of such salts include sodium salts, potassium salts, lysine salts and arginine salts. These salts may be
prepared by methods known to those skilled in the art.
Certain compounds of formulas (I), (IA) and (IB) and their salts may exist in more than one
crystal form and the present invention includes each crystal form and mixtures thereof.
Certain compounds of formulas (I), (IA) and (IB) and their salts may also exist in the form
of solvates, for example hydrates, and the present invention includes each solvate and mixtures
thereof.
Certain compounds of formulas (I), (IA) and (IB) may contain one or more chiral centers,
and exist in different optically active forms. When compounds of formulas (I), (I A) and (IB) contain
one chiral center, the compounds exist in two enantiomeric forms and the present invention includes
both enantiomers and mixtures of enantiomers, such as racemic mixtures. The enantiomers may be
resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric
salts which may be separated, for example, by crystallization; formation of diastereoisomeric
derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or
liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent,
for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral
environment, for example on a chiral support for example silica with a bound chiral Iigand or in the
presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted
into another chemical entity by one of the separation procedures described above, a further step is
required to liberate the desired enantiomeric form. Alternatively, specific enantiomers may be
synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents,
or by converting one enantiomer into the other by asymmetric transformation.
When a compound of formulas (I), (IA) and (IB) contains more than one chiral center it may
exist in diastereoisomeric forms. The diastereoisomeric pairs may be separated by methods known
to those skilled in the art, for example chromatography or crystallization and the individual
enantiomers within each pair may be separated as described above. The present invention includes
each diastereoisomer of compounds of formulas (I), (IA) and (IB) and mixtures thereof.
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Certain compounds of formulas (I), (I A) and (IB) may exist in different tautomeric forms or
as different geometric isomers, and the present invention includes each tautomer and/or geometric
isomer of compounds of formulas (I), (IA) and (IB) and mixtures thereof.
Certain compounds of formulas (I), (IA) and (IB) may exist in different stable
5 conformational forms which may be separable. Torsional asymmetry due to restricted rotation about
an asymmetric single bond, for example because of steric hindrance or ring strain, may permit
separation of different conformers and atropisomers. The present invention includes each
conformational isomer of compounds of formulas (I), (IA) and (IB) and mixtures thereof.
Certain compounds of formulas (I), (IA) and (IB) may exist in zwitterionic form and the
10 present invention includes each zwitterionic form of compounds of formula (I), (I A) and (IB) and
mixtures thereof.
Compounds of formulas (I) 5 (IA) and (IB) include compounds identical to those depicted but
for the fact that one or more hydrogen or carbon atoms are replaced by isotopes thereof. Such
compounds are useful as research and diagnostic tools in metabolism pharmokinetic studies and in
15 binding assays. For example, specific applications in research include radioligand binding assays,
autoradiography studies and in vivo binding studies. Included among the radiolabeled forms of
compounds of the formulas (I), (I A) and (IB) are the tritium and C 14 isotopes thereof.
The compounds of this invention have inhibitory activity against protein kinases. That is,
these compounds modulate signal transduction by protein kinases. Compounds of this invention
20 inhibit protein kinases from serine/threonine and tyrosine kinase classes. In particular, these
compounds selectively inhibit the activity of the KDR/FLK-l/VEGFR-2 and Flt-4/VEGFR-3
tyrosine kinases. Certain compounds of this invention also inhibit the activity of additional tyrosine
kinases such as FIt-l/VEGFR-1, Tie-2 5 Tie-], FGFR, PDGFR, IGF-1R, c-Met, Src-subfamily
kinases such as Lck, Src, fyn, blk, Lyn, yes, etc. Additionally, some compounds of this invention
25 significantly inhibit serine/threonine kinases such as PKC, MAP kinases, erk, CDKs, Plk-1, or Raf-1
which play an essential role in cell proliferation and cell-cycle progression. The potency and
specificity of the generic compounds of this invention towards a particular protein kinase can often
be altered and optimized by variations in the nature, number and arrangement of the substituents
(i.e., W, Rl, R 2 , r3 s y, Q and X*) and conformational restrictions. In addition the metabolites of
30 certain compounds may also possess significant protein kinase inhibitory activity. These metabolite
structures administered alone or generated in vivo may contribute to the observed efficacy.
The compounds of this invention, when administered to individuals in need of such
compounds, inhibit vascular hyperpermeability and the formation of edema in these individuals.
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These compounds act, it is believed, at least in part by inhibiting the activity of KDR tyrosine kinase
which is involved in the process of vascular hyperpermeabil ity and edema formation. The KDR
tyrosine kinase may also be referred to as FLK-1 tyrosine kinase, NYK tyrosine kinase or VEGFR-2
tyrosine kinase. KDR tyrosine kinase is activated when vascular endothelial cell growth factor
5 (VEGF) or another activating ligand (such as VEGF-C, VEGF-D, VEGF-E or HIV Tat protein)
binds to a KDR tyrosine kinase receptor which lies on the surface of vascular endothelial cells.
Following such KDR tyrosine kinase activation, hyperpermeabil ity of the blood vessels occurs and
fluid moves from the blood stream past the blood vessel walls into the interstitial spaces, thereby
forming an area of edema, Diapedesis also often accompanies this response. Similarly, excessive
0 vascular hyperpermeability can disrupt normal molecular exchange across the endothelium in critical
tissues and organs (e.g., brain, lung and kidney), thereby causing macromolecular extravasation and
deposition. Following this acute response to KDR stimulation which is believed to facilitate the
subsequent angiogenic process, prolonged KDR tyrosine kinase stimulation results in the
proliferation and chemotaxis of vascular endothelial cells and formation of new vessels. By
5 inhibiting KDR tyrosine kinase activity, either by blocking the production of the activating ligand,
by blocking the activating ligand binding to the KDR tyrosine kinase receptor, by preventing
receptor dimerization and transphosphorylation, by inhibiting the enzyme activity of the KDR
tyrosine kinase (inhibiting the phosphorylation function of the enzyme) or by some other mechanism
that interrupts its downstream signaling (D. Mukhopedhyay et al } Cancer Res, 55/1278-1284 (1998)
3 and references therein), hyperpermeability, as well as associated extravasation, subsequent edema
formation and matrix deposition, and angiogenic responses, may be inhibited and minimized.
The method of the present invention is useful in the treatment of protein kinase-mediated
conditions, such as any of the conditions described above. In one embodiment, the protein kinase-
mediated condition is characterized by undesired angiogenesis, edema, or stromal deposition. For
5 example, the condition can be one or more ulcers, such as ulcers caused by bacterial or fungal
infections, Mooren ulcers and ulcerative colitis. The condition can also be due to a microbial
infection, such as Lyme disease, sepsis, septic shock or infections by Herpes simplex, Herpes Zoster,
human immunodeficiency virus, protozoa, toxoplasmosis or parapoxvirus; an angiogenic disorders,
such as von Hippel Lindau disease, polycystic kidney disease, pemphigoid, Paget's disease and
) psoriasis; a reproductive condition, such as endometriosis, ovarian hyperstimulation syndrome,
preeclampsia or menometrorrhagia; a fibrotic and edemic condition, such as sarcoidosis, fibrosis,
cirrhosis, thyroiditis, hyperviscosity syndrome systemic, Osier- Weber-Rendu disease, chronic
occlusive pulmonary disease, asthma, and edema following burns, trauma, radiation, stroke, acute
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injury, hypoxia or ischemia; or an inflammatory/immunologic condition, such as systemic lupus,
chronic inflammation, glomerulonephritis, synovitis, inflammatory bowel disease, Crohn's disease,
rheumatoid arthritis, osteoarthritis, multiple sclerosis and graft rejection. Suitable protein kinase-
mediated conditions also include sickle cell anaemia, osteoporosis, osteopetrosis, tumor-induced
5 hypercalcemia and bone metastases. Additional protein kinase-mediated conditions which can be
treated by the method of the present invention include ocular conditions such as ocular and macular
edema, ocular neovascular disease, scleritis, radial keratotomy, uveitis, vitritis, myopia, optic pits,
chronic retinal detachment, post-laser complications, conjunctivitis, Stargardt ? s disease and Eales
disease, in addition to retinopathy and macular degeneration.
10 It has been noted that Streptococcus pneumoniae (pneumococcal infections) stimulate
neutrophil production/secretion of VEGF {Infection & Immunity, 68 (8), 4792-4794 (2000)). VEGF
is also noted to be elevated in cystic fibrosis and correlates with pulmonary exacerbation (Am. J.
Respir. Care Med., 161, 1877-1880 (2000)). Therefore, a compound of the present invention is
useful in treating complications such as pulmonary exacerbation arising from the increased levels of
15 VEGF associated with S. pneumoniae infection or onset of cystic fibrosis.
The compounds of the present invention are also useful in the treatment of cardiovascular
conditions such as atherosclerosis, restenosis, vascular occlusion and carotid obstructive disease.
The compounds of the present invention are also useful in the treatment of cancer related
indications such as various solid tumors, carcinomas, sarcomas (especially Ewing ? s sarcoma and
20 osteosarcoma), retinoblastoma, rhabdomyosarcomas, neuroblastoma, hematopoietic malignancies,
including leukaemia and lymphoma, tumor-induced pleural or pericardial effusions, and malignant
ascites.
Castleman's disease is a lymphoproliferative disorder characterized by enlarged hyperplastic
lymph nodes with marked vascular proliferation. Human IL-6 produced in the affected lymph nodes
25 of Castleman's disease may be responsible for the increased VEGF-production by plasma cells and
vascular proliferation in the lymph node, Nishi, J., and Maryuma, I., Leak Lymphoma, 38 387.
Compounds of the present invention which antagonize VEGF-signaling are useful in the treatment of
Castleman's disease.
The compounds of the present invention are also useful in the treatment of Crow-Fukase
30 (POEMS) syndrome and diabetic conditions such as glaucoma, diabetic retinopathy and
microangiopathy.
One group of preferred compounds of this invention have the property of inhibiting KDR
tyrosine kinase activity without significantly inhibiting Fit- 1 tyrosine kinase activity (Flt-1 tyrosine
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kinase is also referred to as VEGFR-1 tyrosine kinase). Both KDR tyrosine kinase and Fit- 1
tyrosine kinase are activated by VEGF binding to KDR tyrosine kinase receptors and to Fit- 1
tyrosine kinase receptors, respectively. Certain preferred compounds of this invention are unique
because they inhibit the activity of one VEGF-receptor tyrosine kinase (KDR) that is activated by
5 activating ligands but do not inhibit other receptor tyrosine kinases, such as Fit- 1 , that are also
activated by certain activating ligands. In this manner, certain preferred compounds of this invention
are, therefore, selective in their tyrosine kinase inhibitory activity.
In one embodiment, the present invention provides a method of treating a protein kinase-
mediated condition in a patient, comprising administering to the patient a therapeutically or
10 prophylactically effective amount of one or more compounds of Formula I.
A "protein kinase-mediated condition" or a "condition mediated by protein kinase activity" is a
medical condition, such as a disease or other undesirable physical condition, the genesis or
progression of which depends, at least in part, on the activity of at least one protein kinase. The
protein kinase can be, for example, a protein tyrosine kinase or a protein serine/threonine kinase.
15 The patient to be treated can be any animal, and is preferably a mammal, such as a
domesticated animal or a livestock animal. More preferably, the patient is a human.
A "therapeutically effective amount" is an amount of a compound of Formula I or a
combination of two or more such compounds, which inhibits, totally or partially, the progression of
the condition or alleviates, at least partially, one or more symptoms of the condition. A
20 therapeutically effective amount can also be an amount which is prophylactically effective. The
amount which is therapeutically effective will depend upon the patient's size and gender, the
condition to be treated, the severity of the condition and the result sought. For a given patient, a
therapeutically effective amount can be determined by methods known to those of skill in the art.
The Src, Tec, Jak, Map, Csk, NFkB and Syk families of kinases play pivotal roles in the
25 regulation of immune function. The Src family currently includes Fyn, Lck, Fgr, Fes, Lyn, Src, Yrk,
Fyk, Yes, Hck, and Blk. The Syk family is currently understood to include only Zap and Syk. The
TEC family includes Tec, Btk, Rlk and Itk. The Janus family of kinases is involved in the
transduction of growth factor and proinflammatory cytokine signals through a number of receptors.
Although BTK and ITK, members of the Tec family of kinases, play a less well understood role in
30 immunobiology, their modulation by an inhibitor may prove therapeutically beneficial. The Csk
family is currently understood to include Csk and Chk. The kinases RIP, IRAK-1, IRAK-2, NIK,
p38 MAP kinases, Jnk, IKK-1 and IKK-2 are involved in the signal transduction pathways for key
pro-inflammatory cytokines, such as TNF and IL-1. By virtue of their ability to inhibit one or more
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of these kinases, compounds of formula I may function as immunomodulatory agents useful for the
maintenance of allografts, the treatment of autoimmune disorders and treatment of sepsis and septic
shock. Through their ability to regulate the migration or activation of T cells, B-cells, mast cells,
monocytes and neutrophils, these compounds could be used to treat such autoimmune diseases and
5 sepsis. Prevention of transplant rejection, either host versus graft for solid organs or graft versus
host for bone marrow, are limited by the toxicity of currently available immunosuppressive agents
and would benefit from an efficacious drug with improved therapeutic index. Gene targeting
experiments have demonstrated the essential role of Src in the biology of osteoclasts, the cells
responsible for bone resorption. Compounds of formula I, through their ability to regulate Src, may
10 also be useful in the treatment of osteoporosis, osteopetrosis, Paget's disease, tumor- induced
hypercalcemia and in the treatment of bone metastases.
A number of protein kinases have been demonstrated to be protooncogenes. Chromosome
breakage (at the ltk kinase break point on chromosome 5), translocation as in the case of the Abl
gene with BCR (Philadelphia chromosome), truncation in instances such as c-Kit or EGFR, or
15 mutation (e.g., Met) result in the creation of dysregulated proteins converting them from
protooncogene to oncogene products. In other tumors, oncogenesis is driven by-an-autocrine or
paracrine ligand/growth factor receptor interactions. Members of the src-family kinases are typically
involved in downstream signal transduction thereby potentiating the proliferative response or
oncogenesis and themselves may become oncogenic by over-expression or mutation. By inhibiting
20 the protein kinase activity of these proteins the disease process may be disrupted. Vascular
restenosis may involve FGF and/or PDGF - promoted smooth muscle and endothelial cell
proliferation. The ligand stimulation of FGFR, PDGFR, IGF1-R and c-Met/w vivo is proangiogenic,
and potentiates angiogenesis dependent disorders. Inhibition of FGFr, PDGFr , c-Met, or IGF1-R
kinase activities individually or in combination may be an efficacious strategy for inhibiting these
25 phenomena. Thus compounds of formula I which inhibit the kinase activity of normal or aberrant c-
kit, c-met, c-fms, src-family members, EGFr, erbB2, erbB4, BCR-Abl, PDGFr, FGFr, IGF1-R and
other receptor or cytosolic tyrosine kinases may be of value in the treatment of benign and neoplastic
proliferative diseases.
In many pathological conditions (for example, solid primary tumors and metastases,
30 Kaposi's sarcoma, rheumatoid arthritis, blindness due to inappropriate ocular neovascularization,
psoriasis and atherosclerosis) disease progression is contingent upon persistent angiogenesis.
Polypeptide growth factors often produced by the disease tissue or associated inflammatory cells,
and their corresponding endothelial cell specific receptor tyrosine kinases (e.g., KLDR/VEGFR-2, Flt-
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1/VEGFR-I, Tie-2/Tek and Tie-1) are essential for the stimulation of endothelial cell growth,
migration, organization, differentiation and the establishment of the requisite new functional
vasculature. As a result of the vascular permeability factor activity of VEGF in mediating vascular
hyperpermeability, VEGF-stimulation of a VEGFR kinase is also believed to play an important role
5 in the formation of tumor ascites, cerebral and pulmonary edema, pleural and pericardial effusions,
delayed-type hypersensitivity reactions, tissue edema and organ dysfunction following trauma, acute
lung injury (ALI), burns, ischemia, diabetic complications, endometriosis, adult respiratory distress
syndrome (ARDS), post-cardiopulmonary bypass-related hypotension and hyperpermeability, and
ocular edema leading to glaucoma or blindness due to inappropriate neovascularization. In addition
10 to VEGF, recently identified VEGF-C and VEGF-D, and virally-encoded VEGF-E or HIV-Tat
protein can also cause a vascular hyperpermeability response through the stimulation of a VEGFR
kinase. KDR/VEGFR-2 and/or Tie-2 and/or Tie-1 are expressed also in a select population of
hematopoietic stem ceils. Certain members of this population are pfuripotent in nature and can be
stimulated with growth factors to differentiate into endothelial cells and participate in vasculogenetic
15 angiogenic processes. For this reason these have been called Endothelial Progenitor Cells (EPCs) (J.
Clin, hivestig, 103 : 1231-1236 (1999)). In some progenitors, Tie-2 may play a role in their
recruitment, adhesion, regulation and differentiation {Blood , 4317-4326 (1997)). Certain agents
according to formula I capable of blocking the kinase activity of endothelial cell specific kinases
could therefore inhibit disease progression involving these situations.
20 Vascular destabilization by the antagonist ligand of Tie-2 (Ang2) is believed to induce an
unstable "plastic" state in the endothelium. In the presence of high VEGF levels a robust angiogenic
response may result; however, in the absence of VEGF or a VEGF-related stimulus, frank vessel
regression and endothelial apoptosis can occur (Genes and Devel. 13: 1055-1066 (1999)). In an
analogous manner a Tie-2 kinase inhibitor can be proangiogenic or antiangiogenic in the presence or
25 absence of a VEGF-related stimulus, respectively.
The compounds of formula I or a salt thereof or pharmaceutical compositions containing a
therapeutically effective amount thereof may be used in the treatment of protein kinase-mediated
conditions, such as benign and neoplastic proliferative diseases and disorders of the immune system,
as described above. For example, such diseases include autoimmune diseases, such as rheumatoid
30 arthritis, thyroiditis, type 1 diabetes, multiple sclerosis, sarcoidosis, inflammatory bowel disease,
Crohn's disease, myasthenia gravis and systemic lupus erythematosus; psoriasis, organ transplant
rejection (eg. kidney rejection, graft versus host disease), benign and neoplastic proliferative
diseases, human cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate
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and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), and diseases involving
inappropriate vascularization for example diabetic retinopathy, retinopathy of prematurity, choroidal
neovascularization due to age-related macular degeneration, and infantile hemangiomas in human
beinss. In addition, such inhibitors may be useful in the treatment of disorders involving VEGF
mediated edema, ascites, effusions, and exudates, including for example macular edema, cerebral
edema, acute lung injury and adult respiratory distress syndrome (ARDS).
The compounds of the present invention may also be useful in the prophylaxis of the above
diseases.
It is envisaged that the disorders listed above are mediated to a significant extent by protein
tyrosine kinase activity involving the VEGF receptors (e.g. KDR, Fit- 1 and/or Tie-2 and/or Tie-1).
By inhibiting the activity of these receptor tyrosine kinases, the progression of the listed disorders is
inhibited because the angiogenic component of the disease state is severely curtailed. The action of
the compounds of this invention, by their selectivity for specific tyrosine kinases, result in a
minimization of side effects that would occur if less selective tyrosine kinase inhibitors were used.
In another aspect the present invention provides compounds of formula I as defined initially
above for use as medicaments, particularly as inhibitors of protein kinase activity for example
tyrosine kinase activity, serine kinase activity and threonine kinase activity. In yet another aspect
the present invention provides the use of compounds of formula I as defined initially above in the
manufacture of a medicament for use in the inhibition of protein kinase activity.
In this invention, the following definitions are applicable:
"Physiologically acceptable salts" refers to those salts which retain the biological
effectiveness and properties of the free bases and which are obtained by reaction with inorganic
acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid or
organic acids such as sulfonic acid, carboxylic acid, organic phosphoric acid, methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, lactic acid, tartaric acid and the like.
"Alkyl" refers to a saturated aliphatic hydrocarbon, including straight-chain and branched-
chain groups. Preferred straight chain and branched alkyl groups include C]-C8 alkyl groups.
"Alkenyl" refers to an aliphatic hydrocarbon having at least one double bond, including
straight-chain and branched-chain groups. Preferred straight chain and branched alkenyl groups
include C]-Cg alkyl groups.
"Alkynyl" refers to an aliphatic hydrocarbon having at least one triple bond, including
straight-chain and branched-chain groups. Preferred straight chain and branched alkynyl groups
include C]-Cg alkyl groups.
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"Alkoxy" refers to an "O-alkyl" group, where "alkyl" is defined as described above.
"Cycloalkyl" refers to mono-, bi- and tri-carbocyclic groups having 3 to 12 carbon atoms,
preferred cycloalkyl groups have 3 to 6 ring carbon atoms.
"Heterocyclyl" means an optionally substituted mono- or bi-cyclic aromatic or non-aromatic
5 heterocycle in which the heterocycle contains 1, 2, 3 or 4 hetero atoms selected from nitrogen,
sulphur or oxygen. The heterocyclyl group may be attached through a carbon atom or a hetero atom.
Suitable heterocyclyl groups include but are not restricted to 1,3-dioxolanyl, 1,4-dioxolanyl,
morpholinyl, piperidinyl, piperazinyl, thiomorpholinyl, 3H-indolyl, 4H-quinolizinyI, 2-imidazoIinyI,
imidazolidinyl, quinuclidinyl, 2-pyrazolinyl, pyrazolidinyl, 2H~pyranyl, 4H-pyranyl, 1,4-dithianyl,
10 1,3,5-trithianyI, tetrahydrofuranyl, pyrrolidinyl, pyrrolyl, imidazolyl, isothiazolyl, pyrazolyl,
thiazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl,
benzimidazolyl, quinolinyl, isoquinolinyl, indazolyl, furanyl, 2,3,4,5-tetrahydrofuranyl, thienyl,
benzofuranyl, indolizinyl, imidazopyridinyl, isoxazolyl, benzoxazolyl, indolyl, isoindolyU indolinyl,
benzothiazolyl, benzothienyl, purinyl, 1,2,3-triazolyl, 1 ,2,4-trizolyl, 1,3,5-triazinyl, cinnolinyl,
15 phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthypyridinyl, pteridinyl, carbazolyl, acridinyl,
phenazinyl, phenothiazinyl and phenoxazinyl.
"Aryl" means a mono-, bi- or tri-cyclic aromatic group. Suitable aryl groups include phenyl,
indenyl, naphthyl, azulenyl, flourenyl and anthracenyl.
The term "optionally substituted" as used herein refers to substituents that can be attached to
20 moities to which the term refers. Particularly preferred substituents of phenyl, naphthyl and
heterocyclyl groups, which can be substituted by one or more groups, are as follows: a) halo, b) C]_
6 alkyl optionally substituted by one or more of the following: hydroxy, halo, an optionally
substituted amino group or a five, six or seven membered saturated heterocyclic ring containing a
nitrogen atom which optionally contains an additional hetero atom selected from O, S or N and is
25 optionally substituted by a C alkyl group wherein said saturated ring is attached through a carbon
atom c) Ci„6 alkoxy optionally substituted by one or more of the following: hydroxy, Cj^alkoxy,
halo or optionally substituted amino group, or a five, six or seven membered saturated heterocyclic
ring containing a nitrogen atom which optionally contains an additional hetero atom selected from O,
S or N and is optionally substituted by a C|_ 6 alkyl group wherein said saturated ring is attached
30 through a carbon atom d) optionally substituted phenoxy, where the substituents are selected from
the same group of preferred substituents as outlined in this paragraph, e) hydroxy, f)-COR a where
R a is hydroxy, C]„6 alkoxy or -NRbR c , where Rb and R c independently are hydrogen, Ci„]2 alkyl,
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C3-12 cycloalkyl or phenyl wherein the C]. 12 alkyl group, the C2-\2 cycloalkyl group and phenyl
are optionally substituted by one or more of the following: hydroxy, halo, C3„]2 cycloalkyl or-
NRj 1 Rj j wherein Rj, and Rj independently are hydrogen or Cj.g alkyl or wherein R^ and Rj together
with the nitrogen atom to which they are attached is a five, six or seven membered saturated
5 heterocyclic ring which optionally contains an additional hetero atom selected from O, S or N and is
optionally substituted by a Cj.g alkyl group, g) -NR^Re where R<j and R e are each independently
selected from the group consisting of hydrogen, C\.\2 alkyl, cycloalkyl or phenyl or -CORf
wherein Rf is hydrogen, C\„\2 al M, C3-12 cvc 'oalkyl, phenyl-C]„6aIkyl or phenyl wherein in each
case the alkyl group, the cycloalkyl group, phenyl-Cj.galkyl and phenyl are optionally substituted
10 by one or more of the following: halo, hydroxy, nitro or -NRhRj wherein Rh and Rj are
independently selected from the same moieties as defined above, h) -0(CH2) m R g where m is 2, 3, 4
or 5 and Rg is hydroxy or a group of formula -NR^Re where R<j and R e are independently selected
from the same moieties as defined above; or Rg is -COR a wherein R a is independently selected from
the same moieties as defined above and, i) nitro, j) optionally substituted phenyl C]\_6 alkyl, k)
15 optionally substituted phenyl-Ci.galkoxy, 1) cyano, m) Cs.galkenyloxy, n) pyridyloxy or pyridylthio
group wherein the pyridyl ring is optionally substituted by one or more oftrifluoromethyl or nitro, o)
hydroxyamidino, p) aminomethyl, q) formamidomethyl, r) C]_6 alkythio, s) phenyl or t) C2-4
alkenyl or C2-4 alkynyl wherein each is optionally substituted by phenyl which in turn is optionally
substituted by one or more of the following: Cj.g alkyl, Cj_6 alkoxy or halo.
20 Pharmaceutical Formulations
The compounds of this invention can be administered to a human patient by themselves or in
pharmaceutical compositions where they are mixed with suitable carriers or excipient(s) at doses to
treat or ameliorate vascular hyperpermeability, edema, fibrosis, angiogenesis, tumor growth,
psoriasis, arthritis, hyperproliferation and associated disorders. Mixtures of these compounds can
25 also be administered to the patient as a simple mixture or in suitable formulated pharmaceutical
compositions. A therapeutically effective dose further refers to that amount of the compound or
compounds sufficient to result in the prevention or attenuation of inappropriate neovascularization,
progression of hyperproliferative disorders, edema, VEGF-associated hyperpermeability and/or
VEGF-related hypotension. Techniques for formulation and administration of the compounds of the
30 instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co.,
Easton, PA, latest edition.
Routes of Administration
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Suitable routes of administration may, for example, include oral, eyedrop, rectal,
transmucosal, topical, or intestinal administration; parenteral delivery, including intramuscular,
subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous,
intraperitoneal, intranasal, or intraocular injections.
5 Alternatively, one may administer the compound in a local rather than a systemic manner,
for example, via injection of the compound directly into the diseased or an edematous site, often in a
depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system, for example,
in a liposome coated with endothelial cell-specific antibody.
1 0 Composition/Formulation
The pharmaceutical compositions of the present invention may be manufactured in a manner
that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be
15 formulated in conventional manner using one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the active compounds into preparations
which can be used pharmaceutical Iy. Proper formulation is dependent upon the route of
administration chosen.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably
20 in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological
saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated
are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining the active
compounds with pharmaceutical ly acceptable carriers well known in the art. Such carriers enable
25 the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained by combining the active compound with a
solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after
adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in
30 particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
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and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar
solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone,
5 carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings
for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of
gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
10 The push-fit capsules can contain the active ingredients in admixture with filler such as lactose,
binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable
liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may
be added. All formulations for oral administration should be in dosages suitable for such
15 administration.
For buccal administration, the compositions may take the form of tablets or lozenges
formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention
are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a
20 nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered
amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be
formulated containing a powder mix of the compound and a suitable powder base such as lactose or
25 starch.
The compounds can be formulated for parenteral administration by injection, e.g. bolus
injection or continuous infusion. Formulations for injection may be presented in unit dosage form,
e.g.in ampoules or in multi-dose containers, with an added preservative. The compositions may take
such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain
30 formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the
active compounds in water-soluble form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include
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fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or
liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
suspension may also contain suitable stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable
vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other
glycerides.
In addition to the formulations described previously, the compounds may also be formulated
as a depot preparation. Such long acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly or by intramuscular injection). Thus, for example, the
compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for
example, as a sparingly soluble salt.
An example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a
cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic
polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is
a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65%
w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system
(VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent
system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic
administration. Naturally, the proportions of a co-solvent system may be varied considerably
5 without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-
solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be
used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other
biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other
sugars or polysaccharides may substitute for dextrose.
0 Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be
employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for
hydrophobic drugs. Certain organic solvents such as dimethysulfoxide also may be employed,
although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using
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a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers
containing the therapeutic agent. Various sustained-release materials have been established and are
well known by those skilled in the art. Sustained-release capsules may, depending on their chemical
nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical
5 nature and the biological stability of the therapeutic reagent, additional strategies for protein
stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or
excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate,
calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as
10 polyethylene glycols.
Many of the compounds of the invention may be provided as salts with pharmaceutically
compatible counterions. Pharmaceutically compatible salts may be formed with many acids,
(including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.) and
with bases (including but not limited to sodium, potassium, lithium, tetralkylammonia, etc.). Salts
15 tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base
forms.
Effective Dosage
Pharmaceutical compositions suitable for use in the present invention include compositions
wherein the active ingredients are contained in an effective amount to achieve its intended purpose.
20 More specifically, a therapeutically effective amount means an amount effective to prevent
development of or to alleviate the existing symptoms of the subject being treated. Determination of
the effective amounts is well within the capability of those skilled in the art.
For any compound used in the method of the invention, the therapeutically effective dose
can be estimated initially from cellular assays. For example, a dose can be formulated in cellular
25 and animal models to achieve a circulating concentration range that includes the IC50 as determined
in cellular assays (i.e., the concentration of the test compound which achieves a half-maximal
inhibition of a given protein kinase activity). In some cases it is appropriate to determine the IC50 in
the presence of 3 to 5% serum albumin since such a determination approximates the binding effects
of plasma protein on the compound. Such information can be used to more accurately determine
30 useful doses in humans. Further, the most preferred compounds for systemic administration
effectively inhibit protein kinase signaling in intact cells at levels that are safely achievable in
plasma.
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A therapeutically effective dose refers to that amount of the compound that results in
amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for
determining the maximum tolerated dose (MTD) and the ED50 (effective dose for 50% maximal
5 response). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be
expressed as the ratio between MTD and ED50. Compounds which exhibit high therapeutic indices
are preferred. The data obtained from these cell culture assays and animal studies can be used in
formulating a range of dosage for use in humans. The dosage of such compounds lies preferably
within a range of circulating concentrations that include the ED50 with little or no toxicity. The
10 dosage may vary within this range depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See e.g. Fingl et aL, 1975, in "The
Pharmacological Basis of Therapeutics", Ch. 1 pi). In the treatment of crises, the administration of
an acute bolus or an infusion approaching the MTD may be required to obtain a rapid response.
15 Dosage amount and interval may be adjusted individually to provide plasma levels of the
active moiety which are sufficient to maintain the kinase modulating effects, or minimal effective
concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro
data; e.g. the concentration necessary to achieve 50-90% inhibition of protein kinase using the
assays described herein. Dosages necessary to achieve the MEC will depend on individual
20 characteristics and route of administration. However, HPLC assays or bioassays can be used to
determine plasma concentrations.
Dosage intervals can also be determined using the MEC value. Compounds should be
administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time,
preferably between 30-90% and most preferably between 50-90% until the desired amelioration of
25 symptoms is achieved. In cases of local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the subject being
treated, on the subjects weight, the severity of the affliction, the manner of administration and the
judgment of the prescribing physician.
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Packaging
The compositions may, if desired, be presented in a pack or dispenser device which may
contain one or more unit dosage forms containing the active ingredient. The pack may for example
comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be
accompanied by instructions for administration. Compositions comprising a compound of the
invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an
appropriate container, and labeled for treatment of an indicated condition.
in some formulations it may be beneficial to use the compounds of the present invention in
the form of particles of very small size, for example as obtained by fluid energy milling.
The use of compounds of the present invention in the manufacture of pharmaceutical
compositions is illustrated by the following description. In this description the term "active
compound" denotes any compound of the invention but particularly any compound which is the final
product of one of the following Examples.
a) Capsules
In the preparation of capsules, for example, 10 parts by weight of active compound and 240
parts by weight of lactose can be de-aggregated and blended. The mixture can be filled into hard
gelatin capsules, each capsule containing a unit dose or part of a unit dose of active compound.
b) Tablets
Tablets can be prepared from the following ingredients, for example:
Parts by weight
Active compound 10
Lactose 1 90
Maize starch 22
Polyvinylpyrrolidone 1 0
Magnesium stearate 3
The active compound, the lactose and some of the starch can be de-aggregated, blended and
the resulting mixture can be granulated with a solution of the polyvinyl- pyrrolidone in ethanok The
dry granulate can be blended with the magnesium stearate and the rest of the starch. The mixture is
then compressed in a tabletting machine to give tablets each containing a unit dose or a part of a unit
dose of active compound,
c) Enteric coated tablets
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Tablets can be prepared by the method described in (b) above. The tablets can be enteric
coated in a conventional manner using a solution of 20% cellulose acetate phthalate and 3% diethyl
phthalate in ethanokdichloromethane (1:1).
d) Suppositories
In the preparation of suppositories, for example, 100 parts by weight of active compound
can be incorporated in 1300 parts by weight of triglyceride suppository base and the mixture formed
into suppositories each containing a therapeutically effective amount of active ingredient.
In the compositions of the present invention the active compound may, if desired, be
associated with other compatible pharmacologically active ingredients. For example, the compounds
of this invention can be administered in combination with one or more additional pharmaceutical
agents that inhibit or prevent the production of VEGF or angiopoietins, attenuate intracellular
responses to VEGF or angiopoietins, block intracellular signal transduction, inhibit vascular
hyperpermeability, reduce inflammation, or inhibit or prevent the formation of edema or
neovascularization. A Tie-2 inhibitor would be useful in treating pyogenic granuloma of human
<nnsiva (J. Periodont. Res, 2000: 35: 165-171). The compounds of the invention can be administered
prior to, subsequent to or simultaneously with the additional pharmaceutical agent, whichever course
of administration is appropriate. The additional pharmaceutical agents include but are not limited to
anti-edemic steroids, NSAIDS, ras inhibitors, anti-TNF agents, anti-ILl agents, antihistamines, PAF-
antagonists, COX-1 inhibitors, COX-2 inhibitors, NO synthase inhibitors, Akt/PTB inhibitors, IGF-
1R inhibitors, PKC inhibitors, chemotherapy agents such as mitomycin C or Paclitaxel, a vascular
targeting agent such as combretastatin A4, a tubulin binding agent such as a dolastatin, and PI3
kinase inhibitors. The compounds of the invention and the additional pharmaceutical agents act
either additively or synergisticaliy. Thus, the administration of such a combination of substances
that inhibit angiogenesis, vascular hyperpermeability and/or inhibit the formation of edema can
provide greater relief from the deletrious effects of an inflammatory or hyperproliferative disorder,
angiogenesis, vascular hyperpermeability or edema than the administration of either substance alone.
In the treatment of malignant disorders combinations with antiproliferative or cytotoxic
chemotherapies or radiation are anticipated.
The present invention also comprises the use of a compound of formula I as a medicament.
A further aspect of the present invention provides the use of a compound of formula I or a
salt thereof in the manufacture of a medicament for treating vascular hyperpermeability,
angiogenesis-dependent disorders, proliferative diseases and/or disorders of the immune system in
mammals, particularly human beings.
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The present invention also provides a method of treating vascular hyperpermeability,
inappropriate neovascularization, proliferative diseases and/or disorders of the immune system
which comprises the administration of a therapeutically effective amount of a compound of formula
I to a mammal, particularly a human being, in need thereof.
5 The in vitro potency of compounds in inhibiting these protein kinases may be determined by
the procedures detailed below.
The potency of compounds can be determined by the amount of inhibition of the
phosphorylation of an exogenous substrate (e.g., synthetic peptide (Z. Songyang et aL, Nature.
373:536-539) by a test compound relative to control.
10 KDR Tyrosine Kinase Production Using Baculovirus System:
The coding sequence for the human KDR intra-cellular domain (aa789-1354) was generated
through PCR using cDNAs isolated from HUVEC cells. A poly-His6 sequence was introduced at
the N-terminus of this protein as well. This fragment was cloned into transfection vector pVL1393
at the Xba 1 and Not 1 site. Recombinant baculovirus (BV) was generated through co-transfection
15 using the BaculoGold Transfection reagent (PharMingen). Recombinant BV was plaque purified
and verified through Western analysis. For protein production, SF-9 cells were grown in SF-900-II
medium at 2 x 106/ml, and were infected at 0.5 plaque forming units per cell (MOI). Cells were
harvested at 48 hours post infection.
Purification of KDR
20 SF-9 cells expressing (His)6KDR(aa789-1354) were lysed by adding 50 ml of Triton X-100
lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, ImM PMSF,
10p.g/ml aprotinin, 1 \xg/m\ leupeptin) to the cell pellet from 1L of cell culture. The lysate was
centrifuged at 19,000 rpm in a Sorvai SS-34 rotor for 30 min at 4EC. The cell lysate was applied to
a 5 ml N1CI2 chelating sepharose column, equilibrated with 50 mM HEPES, pH7.5, 0.3 M NaCI.
25 KDR was eluted using the same buffer containing 0.25 M imidazole. Column fractions were
analyzed using SDS-PAGE and an ELISA assay (below) which measures kinase activity. The
purified KDR was exchanged into 25mM HEPES, pH7.5, 25mM NaCl, 5 mM DTT buffer and
stored at -80EC.
Human Tie-2 Kinase Production and Purification
30 The coding sequence for the human Tie-2 intra-cellular domain (aa775-l 124) was generated
through PCR using cDNAs isolated from human placenta as a template. A poly-Hisg sequence was
introduced at the N-terminus and this construct was cloned into transfection vector pVL 1939 at the
Xba 1 and Not 1 site. Recombinant BV was generated through co-transfection using the BaculoGold
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10
Transfection reagent (PharMingen). Recombinant BV was plaque purified and verified through
Western analysis. For protein production, SF-9 insect cells were grown in SF-900-II medium at 2 x
106/ml, and were infected at MOI of 0.5. Purification of the His-tagged kinase used in screenino
was analogous to that described for KDR.
Human Fit- 1 Tyrosine Kinase Production and Purification
The baculoviral expression vector pVL1393 (Phar Mingen, Los Angeles, CA) was used. A
nucleotide sequence encoding poly-His6 was placed 5' to the nucleotide region encoding the entire
intracellular kinase domain of human Flt-1 (amino acids 786-1338). The nucleotide sequence
encoding the kinase domain was generated through PCR using cDNA libraries isolated from
HUVEC cells. The histidine residues enabled affinity purification of the protein as a manner
analogous to that for KDR and ZAP70. SF-9 insect cells were infected at a 0.5 multiplicity and
harvested 48 hours post infection.
Flt-4 VEGF3 baculovirus expression vector
The cDNA corresponding to the intracellular kinase domain of human Flt-4/VEGF 3 was
15 obtained by performing Polymerase Chain Reaction (PCR) with Flt-4 specific oligonucleotide
primers on total cDNA from human placenta (Galland, F. et.aL, Oncogene (1993), 8, 1233-1240).
The cDNA sequence was compared and verified using the published GENBANK sequence
(x69878.gb_prl). FLT-4 cDNA sequence corresponding to bp. 2413-3918 ie. aa. Cys798-Argl298
was subcloned into the baculovirus expression vector pFastbacB (Life Technologies). The
20 subcloning introduced 4 extra amino acids Ala-Met-Gly-Ser in front of Cys798. Therefore,, the
fusion protein produced would have Met-(His)g, a spacer region, a Tev protease cleavage site
followed by Flt-4 kinase domain including the 4 extra amino acids.. The expression vector was
introduced into SF9 cells to produce baculovirus which was then used to infect more SF9 cells and
express Flt-4/VEGF3 kinase domain protein.
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EGFR Tyrosine Kinase Source
EGFR was purchased from Sigma (Cat # E-3641; 500 units/50 and the EGF ligand was
acquired from Oncogene Research Products/Calbiochem (Cat # PF01 1-100).
Expression of ZAP70
The baculoviral expression vector used was pVL1393. (Pharmingen, Los Angeles, Ca.) The
nucleotide sequence encoding amino acids M(H)6 LVPR9S was placed 5' to the region encoding the
entirety of ZAP70 (amino acids 1-619). The nucleotide sequence encoding the ZAP70 coding region
was generated through PGR using cDNA libraries isolated from Jurkat immortalized T-cells. The
histidine residues enabled affinity purification of the protein (vide infra). The LVPR9S bridge
constitutes a recognition sequence for proteolytic cleavage by thrombin, enabling removal of the
affinity tag from the enzyme. SF-9 insect cells were infected at a multiplicity of infection of 0.5 and
harvested 48 hours post infection.
Extraction and purification of ZAP70:
SF-9 cells were lysed in a buffer consisting of 20 mM Tris, pH 8.0, 137 mM NaCI, 10%
glycerol, 1% Triton X-100, 1 mM PMSF, 1 jig/ml leupeptin, 10 ]-ig/ml aprotinin and 1 mM sodium
orthovanadate. The soluble lysate was applied to a chelating sepharose HiTrap column (Pharmacia)
equilibrated in 50 mM HEPES, pH 7.5, 0.3 M NaCI. Fusion protein was eluted with 250 mM
imidazole. The enzyme was stored in buffer containing 50 mM HEPES, pH 7.5, 50 mM NaCI and 5
mM DTT.
Protein kinase source:
Lck, Fyn, Src, BIk, Csk, and Lyn, and truncated forms thereof may be commercially
obtained ( e.g. from Upstate Biotechnology Inc., Saranac Lake, NY; and Santa Cruz Biotechnology
Inc., Santa Cruz, CA) or purified from known natural or recombinant sources using conventional
methods.
Enzyme Linked Immunosorbent Assay (ELISA) For PTKs:
Enzyme linked immunosorbent assays (ELISA) were used to detect and measure the
presence of tyrosine kinase activity. The ELISA were conducted according to known protocols
which are described in, for example, Voller, et aL, 1980, "Enzyme-Linked Immunosorbent Assay,"
In: Manual of Clinical Immunology, 2d ed, edited by Rose and Friedman, pp 359-371 Am. Soc. of
Microbiology, Washington, D.C.
The disclosed protocol was adapted for determining activity with respect to a specific PTK.
For example, preferred protocols for conducting the ELISA experiments is provided below.
Adaptation of these protocols for determining a compound's activity for other members of the
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receptor PTK family, as well as non-receptor tyrosine kinases, are well within the abilities of those
in the art. For purposes of determining inhibitor selectivity, a universal PTK substrate (e.g., random
copolymer of poly(Glu4 Tyr), 20,000-50,000 MW) was employed together with ATP (typically 5
jj.M) at concentrations approximately twice the apparent Km in the assay.
5 The following procedure was used or can be used to assay the inhibitory effect of
compounds of this invention on KDR, VEGFR-3, Flt-1, Tie-2,Tie-l, EGFR, FGFR, PDGFR, IGF-1-
R 5 Insulin receptor, c-Met, Lck, Blk, Csk, Src, Lyn, Fyn and ZAP70 tyrosine kinase activity:
Buffers and Solutions:
PGTPoly (Glu,Tyr)4:l
10 Store powder at -20°C. Dissolve powder in phosphate buffered saline (PBS) for 50mg/ml solution.
Store 1ml aliquots at -20°C. When making plates dilute to 250^g/ml in Gibco PBS.
Reaction Buffer: lOOmM Hepes, 20mM MgCl2, 4mM MnCl2, 5mM DTT, 0.02%BSA, 200p,M
NaV04, pH 7.10
ATP: Store aliquots of 1 OOmM at -20°C. Dilute to 20pM in water
15 Washing Buffer: PBS with 0.1% Tween 20
Antibody Diluting Buffer: 0.1% bovine serum albumin (BSA) in PBS
TMB Substrate: mix TMB substrate and Peroxide solutions 9:1 just before use or use K-Blue
Substrate from Neogen
Stop Solution: 1M Phosphoric Acid
20 Procedure
1 . Plate Preparation:
Dilute PGT stock (50mg/ml, frozen) in PBS to a 250|ig/ml. Add 125|il per well of Corning
modified flat bottom high affinity ELISA plates (Corning #25805-96). Add 125jil PBS to blank
wells. Cover with sealing tape and incubate overnight 37°C. Wash lx with 250jo.l washing buffer
25 and dry for about 2hrs in 37°C dry incubator.
Store coated plates in sealed bag at 4°C until used.
2. Tyrosine Kinase Reaction:
-Prepare inhibitor solutions at a 4x concentration in 20% DMSO in water.
-Prepare reaction buffer
30 -Prepare enzyme solution so that desired units are in 50p.l, e.g. for KDR make to 1 ng/p.1 for a total
of 50ng per well in the reactions. Store on ice.
-Make 4x ATP solution to 20p,M from lOOmM stock in water. Store on ice
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-Add 50fxl of the enzyme solution per well (typically 5-50 ng enzyme/well depending on the specific
activity of the kinase)
-Add 25|Lil 4x inhibitor
-Add 25JJ.I 4x ATP for inhibitor assay
5 -Incubate for 1 0 minutes at room temperature
-Stop reaction by adding 50}il 0.05N HC1 per well
-Wash plate
**Final Concentrations for Reaction: 5pM ATP, 5% DMSO
3. Antibody Binding
10 -Dilute lmg/ml aliquot of PY20-HRP (Pierce) antibody(a phosphotyrosine antibody)to 50ng/ml in
0.1% BSA in PBS by a 2 step dilution (lOOx, then 200x)
-Add lOOp.1 Ab per well. Incubate 1 hr at room temp. Incubate Ihr at 4C.
-Wash 4x plate
4. Color reaction
15 -Prepare TMB substrate and add 100p.l per well
-Monitor OD at 650nm until 0.6 is reached
-Stop with 1M Phosphoric acid. Shake on plate reader.
-Read OD immediately at 450nm
Optimal incubation times and enzyme reaction , conditions vary slightly with enzyme
20 preparations and are determined empirically for each lot.
For Lck, the Reaction Buffer utilized was 100 mM MOPSO, pH 6.5, 4 mM MnCl2> 20 mM
MgCK 5 mM DTT, 0.2% BSA, 200 mM NaV04 under the analogous assay conditions.
Compounds of formula I may have therapeutic utility in the treatment of diseases involving
both identified, including those not mentioned herein, and as yet unidentified protein tyrosine
25 kinases which are inhibited by compounds of formula I. All compounds exemplified herein
significantly inhibit either FGFR, PDGFR, KDR, VEGFR-3, Tie-2,Tie-I, Lck, Fyn, Blk, Lyn or Src
at concentrations of 50 micromolar or below. Some compounds of this invention also significantly
inhibit other tyrosine or serine/threonine kinases such as cdc2 (cdkl) or Plk-1 at concentrations of 50
micromolar or below.
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Cdc2 source
The human recombinant enzyme and assay buffer may be obtained commercially (New
England Biolabs, Beverly, MA. USA) or purified from known natural or recombinant sources using
conventional methods.
5 Cdc2 Assay
The protocol used was that provided with the purchased reagents with minor modifications.
In brief, the reaction was carried out in a buffer consisting of 50mM Tris pH 7.5, lOOmM NaCl,
ImM EGTA, 2mM DTT, 0.01% Brij, 5% DMSO and lOmM MgCl 2 (commercial buffer)
supplemented with fresh 300 jaM ATP (31 p.Ci/ml) and 30 jug/ml histone type IIIss final
10 concentrations. A reaction volume of 80|iL, containing units of enzyme, was run for 20 minutes at
25 degrees C in the presence or absence of inhibitor. The reaction was terminated by the addition of
\20\xL of 10% acetic acid. The substrate was separated from unincorporated label by spotting the
mixture on phosphocellulose paper, followed by 3 washes of 5 minutes each with 75mM phosphoric
acid. Counts were measured by a betacounter in the presence of liquid scintillant.
15 Certain compounds of this invention significantly inhibit cdc2 at concentrations below 50 uM.
PKC kinase source
The catalytic subunit of PKC may be obtained commercially (Calbiochem).
PKC kinase assay
A radioactive kinase assay was employed following a published procedure (Yasuda, I.,
20 Kirshimoto, A., Tanaka, S., Tominaga, M., Sakurai, A., Nishizuka, Y. Biochemical and Biophysical
Research Communication 3:166, 1220-1227 (1990)). Briefly, all reactions were performed in a
kinase buffer consisting of 50 mM Tris-HCL pH7.5, lOmM MgCl 2 , 2mM DTT, ImM EGTA, 100
\iM ATP, 8 pM peptide, 5% DMSO and 33 p ATP (8Ci/mM). Compound and enzyme were mixed
in the reaction vessel and the reaction initiated by addition of the ATP and substrate mixture.
25 Following termination of the reaction by the addition of 10 p.L stop buffer (5 mM ATP in 75mM
phosphoric acid), a portion of the mixture was spotted on phosphocellulose filters. The spotted
samples were washed 3 times in 75 mM phosphoric acid at room temperature for 5 to 15 minutes.
Incorporation of radiolabel was quantified by liquid scintillation counting.
Erk2 enzyme source
30 The recombinant murine enzyme and assay buffer may be obtained commercially (New
England Biolabs, Beverly MA. USA) or purified from known natural or recombinant sources using
conventional methods.
Erk2 enzyme assay
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In brief, the reaction was carried out in a buffer consisting of 50 mM Tris pH 7.5, ImM
EGTA, 2mM DTT, 0.01% Brij, 5% DMSO and 10 mM MgCl2 (commercial buffer) supplemented
with fresh 100 uM ATP (31 uCi/ml) and 30p.M myelin basic protein under conditions recommended
by the supplier. Reaction volumes and method of assaying incorporated radioactivity were as
described for the PKC assay (vide supra).
In Vitro Models for T-cell Activation
Upon activation by mitogen or antigen, T-cells are induced to secrete 1L-2, a growth factor
that supports their subsequent proliferative phase. Therefore, one may measure either production of
IL-2 from or cell proliferation of, primary T-cells or appropriate T-cell lines as a surrogate for T-cell
activation. Both of these assays are well described in the literature and their parameters well
documented (in Current Protocols in Immunology, Vol 2, 7.10.1-7.1 1.2).
In brief, T-cells may be activated by co-culture with allogenic stimulator cells, a process
termed the one-way mixed lymphophocyte reaction. Responder and stimulator peripheral blood
mononuclear cells are purified by Ficoll-Hypaque gradient (Pharmacia) per directions of the ■
manufacturer. Stimulator cells are mitotically inactivated by treatment with mitomycin C (Sigma) or
gamma irradiation. Responder and stimulator cells are co-cultured at a ratio of two to one in the
presence or absence of the test compound. Typically 1()5 responders are mixed with 5 x 10*
stimulators and plated (200 u.1 volume) in a U bottom microliter plate (Costar Scientific). The cells
are cultured in RPMI 1640 supplemented with either heat inactivated fetal bovine serum (Hyclone
Laboratories) or pooled human AB serum from male donors, 5 x 10"5 M 2mercaptoethanol and
0.5% DMSO, The cultures are pulsed with 0.5 |aCi of 3H thymidine (Amersham) one day prior to
harvest (typically day three). The cultures are harvested (Betaplate harvester, Wallac) and isotope
uptake assessed by liquid scintillation (Betaplate, Wallac).
The same culture system may be used for assessing T-cell activation by measurement of
IL-2 production. Eighteen to twenty-four hours after culture initiation, the supernatants are removed
and the IL-2 concentration is measured by ELISA (R and D Systems) following the directions of the
manufacturer.
In-vivo Models of T-Cell Activation
The in vivo efficacy of compounds can be tested in animal models known to directly
measure T-cell activation or for which T-cells have been proven the effectors. T-cells can be
activated in vivo by ligation of the constant portion of the T-cell receptor with a monoclonal
anti-CD3 antibody (Ab). In this model, BALB/c mice are given 10u.g of anti-CD3 Ab
intraperitoneally two hours prior to exsanguination. Animals to receive a test drug are pre-treated
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with a single dose of the compound one hour prior to anti-CD3 Ab administration. Serum levels of
the proinflammatory cytokines interferon-y (IFN-y) and tumor necrosis factor-a(TNF-a), indicators
of T-cell activation, are measured by ELISA. A similar model employs hi vivo T-cell priming with a
specific antigen such as keyhole limpet hemocyanin (KLH) followed by a secondary in vitro
challenge of draining lymph node cells with the same antigen. As previously, measurement of
cytokine production is used to assess the activation state of the cultured cells. Briefly, C57BL/6 mice
are immunized subcutaneously with 100 jag KLH emulsified in complete Freund T s adjuvant (CFA)
on day zero. Animals are pre-treated with the compound one day prior to immunization and
subsequently on days one, two and three post immunization. Draining lymph nodes are harvested on
day 4 and their cells cultured at 6 x 10 6 per ml in tissue culture medium (RPMI 1640 supplemented
with heat inactivated fetal bovine serum (Hyclone Laboratories) 5 x 10" 5 M 2-mercaptoethanol and
0.5% DMSO) for both twenty- four and forty-eight hours. Culture supernatants are then assessed for
the autocrine T-cell growth factor Interleukin-2 (IL-2) and/or IFN-y levels by ELISA.
Lead compounds can also be tested in animal models of human disease. These are
exemplified by experimental auto-immune encephalomyelitis (EAE) and collagen-induced arthritis
(CIA). EAE models which mimic aspects of human multiple sclerosis have been described in both
rats and mice (reviewed FASEB J. 5:2560-2566, 1991; murine model: Lab. Invest. 4(3):278, 1981;
rodent modehJ. Immunol 146(4): 1 163-8, 1991 ). Briefly, mice or rats are immunized with an
emulsion of myelin basic protein (MBP), or neurogenic peptide derivatives thereof, and CFA. Acute
disease can be induced with the addition of bacterial toxins such as bordetella pertussis.
Relapsing/remitting disease is induced by adoptive transfer of T-cells from MBP/ peptide
immunized animals.
CIA may be induced in DBA/1 mice by immunization with type II collagen (J.
Immunol:142(7):2237-2243). Mice will develop signs of arthritis as early as ten days following
antigen challenge and may be scored for as long as ninety days after immunization. In both the EAE
and CIA models, a compound may be administered either prophylactically or at the time of disease
onset. Efficacious drugs should reduce severity and/or incidence.
Certain compounds of this invention which inhibit one or more angiogenic receptor PTK,
and/or a protein kinase such as lck involved in mediating inflammatory responses can reduce the
0 severity and incidence of arthritis in these models.
Compounds can also be tested in mouse allograft models, either skin (reviewed in Ann. Rev.
Immunol., 10:333-58, 1992; Transplantation: 57(12): 1701-17D6, 1994) or heart
(Am.J.Anat.:! 13:273, 1963). Briefly, full thickness skin grafts are transplanted from C57BL/6 mice
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to BALB/c mice. The grafts can be examined daily, beginning at day six, for evidence of rejection.
In the mouse neonatal heart transplant model, neonatal hearts are ectopically transplanted from
C57BL/6 mice into the ear pinnae of adult CBA/J mice. Hearts start to beat four to seven days post
transplantation and rejection may be assessed visually using a dissecting microscope to look for
5 cessation of beating.
Cellular Receptor PTK Assays
The following cellular assay was used to determine the level of activity and effect of the
different compounds of the present invention on KDR/VEGFR2. Similar receptor PTK assays
employing a specific ligand stimulus can be designed along the same lines for other tyrosine kinases
10 using techniques well known in the art.
VEGF-Induced KDR Phosphorylation in Human Umbilical Vein Endothelial Cells
(HUVEC) as Measured by Western Blots:
1. HUVEC cells (from pooled donors) were purchased from Clonetics (San Diego,
CA) and cultured according to the manufacturer directions. Only early passages (3-8) were used for
15 this assay. Cells were cultured in 100 mm dishes (Falcon for tissue culture; Becton Dickinson;
Plymouth, England) using complete EBM media (Clonetics).
2. For evaluating a compound's inhibitory activity, cells were trypsinized and seeded at
0.5-1.0 x 10 5 cells/well in each well of 6-well cluster plates (Costar; Cambridge, MA).
3. 3-4 days after seeding, plates were 90-100% confluent. Medium was removed from
20 all the wells, cells were rinsed with 5-10ml of PBS and incubated 18-24h with 5ml of EBM base
media with no supplements added (i.e., serum starvation).
4. Serial dilutions of inhibitors were added in 1ml of EBM media (25jjM, 5\xM, or
ljuM final concentration to cells and incubated for one hour at 37 C. Human recombinant
VEGF]65 (R&D Systems) was then added to all the wells in 2 ml of EBM medium at a final
25 concentration of 50ng/ml and incubated at 37 C for 10 minutes. Control cells untreated or treated
with VEGF only were used to assess background phosphorylation and phosphorylation induction by
VEGF.
All wells were then rinsed with 5- 10ml of cold PBS containing ImM Sodium Orthovanadate
(Sigma) and cells were lysed and scraped in 200)^1 of RIPA buffer (50mM Tris-HCl) pH7, 350mM
30 NaCl, 1% NP-40, 0.25% sodium deoxycholate, ImM EDTA) containing protease inhibitors (PMSF
ImM, aprotinin l]ag/ml, pepstatin l|ig/ml, leupeptin ljig/ml, Na vanadate ImM, Na fluoride ImM)
and Ifig/ml of Dnase (all chemicals from Sigma Chemical Company, St Louis, MO). The lysate was
spun at 14,000 rpm for 30min, to eliminate nuclei.
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Equal amounts of proteins were then precipitated by addition of cold (-20 C) Ethanol (2
volumes) for a minimum of 1 hour or a maximum of overnight. Pellets were reconstituted in Laemli
sample buffer containing 5% -mercaptoethanol (BioRad; Hercules, CA) and boiled for 5min. The
proteins were resolved by polyacrylamide gel electrophoresis (PAGE; 6%, 1.5mm Novex, San
5 Deigo, CA) and transferred onto a nitrocellulose membrane using the Novex system. After blocking
with bovine serum albumin (3%), the proteins were probed overnight with anti-KDR polyclonal
antibody (C20, Santa Cruz Biotechnology; Santa Cruz, CA) or with anti-phosphotyrosine
monoclonal antibody (4G10, Upstate Biotechnology, Lake Placid, NY) at 4° C. After washing and
incubating for 1 hour with HRP-conjugated F(ab)2 of goat anti-rabbit or goat-anti-mouse IgG the
10 bands were visualized using 'the emission chemiluminescience (ECL) system (Amersham Life
Sciences, Arlington Height, IL).
Certain examples of the present invention significantly inhibit cellular VEGF-induced KDR
tyrosine kinase phosphorylation at concentrations of less than 50 juM.
The inhibition of VEGF-induced KDR-receptor autophosphorylation can be confirmed with
15 a further in vitro experiment transfected CHO cells (CHO = Chinese hamster ovary), which
permanently express human VEGF receptor (KDR). Cells are seeded in culture medium (with 10%
fetal calf serum = FCS) in 6-well cell-culture plates and incubated at 37°C under 5% CO2 until they
show about 80% confluency. The compounds to be tested are then diluted in culture medium
(without FCS, with 0.1% bovine serum albumin) and added to the cells. (Controls compromise
20 medium without test compounds). After two hours' incubation at 37°C, recombinant VEGF is
added; the final VEGF concentration's 20 ng/ml.) After a further 5 minutes' incubation at 37°C, the
cells are washed twice with ice-cold PBS (phosphate-buffered saline) and immediately lysed in 100
p.1 lysis buffer per well. The lysates are then centrifuged to remove the cell nuclei, and the protein
concentrations of the supernatants are determined using a commercial protein assay (BIORAD).
25 The lysates can then either be immediately used or, if necessary, stored at -20°C before being
assessed using PAGE and immunoblotting as described above.
In vivo Uterine Edema Model
This assay measures the capacity of compounds to inhibit the acute increase in uterine
weight in mice which occurs in the first few hours following estrogen stimulation. This early onset
30 of uterine weight increase is known to be due to edema caused by increased permeability of uterine
vasculature. Cullinan-Bove and Koss (Endocrinology (1993), 735:829-837) demonstrated a close
temporal relationship of estrogen-stimulated uterine edema with increased expression of VEGF
mRNA in the rat uterus. These results have been confirmed by the use of neutralizing monoclonal
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antibody to VEGF which significantly reduced the acute increase in uterine weight following
estrogen stimulation (WO 97/42187). Hence, this system can serve as a model for/77 vivo inhibition
of VEGF signalling and the associated hyperpermeability and edema.
Materials: All hormones were purchased from Sigma (St. Louis, MO) or Cal Biochem (La
5 Jolla, CA) as lyophilized powders and prepared according to supplier instructions.
Vehicle components (DMSO, Cremaphor EL) were purchased from Sigma (St. Louis, MO).
Mice (Balb/c, 8-12 weeks old) were purchased from Taconic (Germantown, NY) and housed in a
pathogen-free animal facility in accordance with institutional Animal Care and Use Committee
Guidelines.
10 Method:
Day 1: Balb/c mice were given an intraperitoneal (i.p.) injection of 12.5 units of
pregnant mare's serum gonadotropin (PMSG).
Day 3: Mice received 15 units of human chorionic gonadotropin (hCG) i.p.
Day 4: Mice were randomized and divided into groups of 5-10. Test compounds
15 were administered by i.p., i.v. or p.o. routes depending on solubility and vehicle at doses ranging
from 1-100 mg/kg. Vehicle control group received vehicle only and two groups were left untreated.
Thirty minutes later, experimental, vehicle and 1 of the untreated groups were given an i.p.
injection of 17 -estradiol (500 mg/kg). After 2-3 hours, the animals were sacrificed by CO2
inhalation. Following a midline incision, each uterus was isolated and removed by cutting just
20 below the cervix and at the junctions of the uterus and oviducts. Fat and connective tissue were
removed with care not to disturb the integrity of the uterus prior to weighing (wet weight). Uteri
were blotted to remove fluid by pressing between two sheets of filter paper with a one liter glass
bottle filled with water. Uteri were weighed following blotting (blotted weight). The difference
between wet and blotted weights was taken as the fluid content of the uterus. Mean fluid content of
25 treated groups was compared to untreated or vehicle treated groups. Significance was determined by
Student's test. Non-stimulated control group was used to monitor estradiol response.
Results demonstrate that certain compounds of the present invention inhibit the formation of
edema when administered systemically by various routes.
Certain compounds of this invention which are inhibitors of angiogenic receptor tyrosine
30 kinases can also be shown active in a Matrigel implant model of neovascularization. The Matrigel
neovascularization model involves the formation of new blood vessels within a clear marble of
extracellular matrix implanted subcutaneously which is induced by the presence of proangiogenic
factor producing tumor cells (for examples see: Passaniti, A., et ah Lab. Investig. (1992), 67(4), 519-
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528; Anat. Rec. (1997), 249(1), 63-73; Int. J. Cancer (1995), 63(5), 694-701; Vase. Biol. (1995),
15(11), 1857-6). The model preferably runs over 3-4days and endpoints include macroscopic
visual/image scoring of neovascularization, microscopic microvessel density determinations, and
hemoglobin quantitation (Drabkin method) following removal of the implant versus controls from
5 animals untreated with inhibitors. The model may alternatively employ bFGF or HGF as the
stimulus.
Certain compounds of this invention which inhibit one or more oncogenic protooncogenic,
or proliferation-dependent protein kinases, or angiogenic receptor PTK also inhibit the growth of
primary murine, rat or human xenograft tumors in mice, or inhibit metastasis in murine models.
10 The antitumor efficacy of a compound of the present invention can be demonstrated in vivo
as follows: in vivo activity in the nude mouse xenotransplant model: female BALB/c nude mice(8-
12 weeks old, for example Novartis Animal Farm, Sisseln, Switzerland) are kept under sterile
conditions with water and feed ad libitum. Tumours are induced by subcutaneous injection of
tumour cells (e.g., human epithelial cell line A-431; American Type Culture Collection (ATCC),
15 Rockville, MD, USA, Catalogue Number ATCC CRL 1555; cell line from 85-year-old woman;
epidermoid carcinoma cell line) into carrier mice. The resulting tumours pass through at least three
consecutive transplantations before the start of treatment if tumor fragments are employed. Tumour
fragments (about 25 mg) are implanted subcutaneously in the left flank of the animals using a 13-
gauge trocar needle under Forene® anaesthesia (Abbott, Switzerland). Treatment with the test
20 compound is started as soon as the tumour has reached a mean volume of 100 mm^. Tumour
growth is measured two to three times a week and 24 hours after the last treatment by determining
the length of two perpendicular axes. The tumour volumes are calculated in accordance with
published methods (see Evans et al. 5 Brit. J. Cancer 45, 466-8 [1982]). The antitumour efficacy is
determined as the mean increase in tumour volume of the treated animals divided by the mean
25 increase in tumour volume of the untreated animals (controls) and, after multiplication by 100, is
expressed as T/C%. Tumour regression (given in %) is reported as the smallest mean tumour
volume in relation to the mean tumour volume at the start of treatment. The test compound is
administered daily by gavage.
As an alternative to cell line A-43 1 , other cell lines may also be used in the same manner, for
30 example:
the MCF-7 breast adenocarcinoma cell line (ATCC No. HTB 22; see also J. Natl. Cancer Inst.
(Bethesda) 51,1 409- 1 6 [ 1 973]);
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the MDA-MB 468 breast adenocarcinoma cell line (ATCC No. HTB 332; see also In Vitro 14,
911-15[1978]);
the MDA-MB 23 1 breast adenocarcinoma cell line (ATTC No. HTB 26; see also J. Natl. Cancer
Inst. (Bethesda) 53, 661-71 [1974]);
5 - the colo 205 colon carcinoma cell line (ATCC No. CCL 222; see also Cancer Res._38, 1345-55
[1978]);
- the HCT 1 16 colon carcinoma cell line (ATCC No. 247; see also Cancer Res. 41, 1751-6 [1981];
- the DU145 prostate carcinoma cell line DU 145 (ATCC No. HTB _81 ; see also Cancer Res. 37,
4049-58 [1978]; or
10 the PC-3 prostate carcinoma cell line PC-3 (ATCC No. CRL 1435; see also Cancer Res. 40, 524-
34 [1980].
The activity of compounds of a compound of the present invention against pain can be
shown in the following mode of nociception (pain). In this model, the hyperalgesia caused by an
inter-planar yeast injection is measured by applying increased pressure to the foot until the animal
15 vocalizes or withdraws its foot from the applied pressure pad. The model is sensitive to COX
inhibitors, diclofenac at 3 mg/kg is used as a positive control.
Method: The baseline pressure required to induce vocalization or withdrawl of the paw of
male Sprague Dawley rats (weighing approximately 180 g, supplied by Iffa Credo, France) is
measured (2 hours before treatment), followed by an intra-planar injection of 100|j.l of a 20% yeast
20 suspension in water in the hind paw. The rats are treated orally with the test compound (3, 10 or 30
mg/kg), diclofenac (3 mg/kg) or vehicle (saline) p.o. 2 hours later (time point 0 hours), and the
pressure test is repeated 1 and 2 hours after dosing. Using the standard apparatus supplied by Ugo
Basile, Italy, the pressure required to induce vocalization or paw withdrawl of the compound-treated
rats at these time points is compared to that of vehicle-treated animals.
25 A test compound of the formula 1 inhibits paw hyperalgesia both at 1 and 2 hours after
dosing in the Randal 1-Selitto test preferably in the 20-75 mg/kg p.o. dose range, preferably by 10 to
100%, demonstrating that the compound has analgesic activity.
On the basis of these studies, a compound of the present invention surprisingly is
appropriate for the treatment of inflammatory (especially rheumatic or rheumatoid) diseases and/or
30 pain.
General Procedures. The compounds of the present invention can be and were synthesized
according to the following description and examples. Unless otherwise specified, all starting
materials and solvents were obtained from commercially available sources and were used without
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further purification. LCMS analyses and purification were performed using a Gilson HPLC system
equipped with a 215 autosampler attached to a Micromass Platform Mass Spectrometer. Acetonitrile
and aqueous 50 mM ammonium acetate (pH 4.5) were used to elute products from either a
Pecosphere CI 8, 3 jam, 33x4.6 mm column or a Hypersil BDS-C18, 5 p.m, 100x20 mm column for
5 analytical or preparative work, respectively. A linear gradient from 0-100% acetonitrile over 4.5
min with a flow rate of 3.5 mL/min was used for analytical analysis. A linear gradient from 0-
100% acetonitrile over 8.5 min with a flow rate of 25 mL/min was used for preparative separations.
NMR spectra were recorded on a Bruker 400 MHz spectrometer with a deuterated solvent as the
internal lock. NMR data are reported as chemical shift (ppm% multiplicity, number of hydrogens,
1 0 where the chemical shift is referenced to TMS.
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Scheme I
W = N0 2 , CI
Scheme II
Scheme III
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Scheme II1A
1C
1. CH 3 i, K + t-BuO-
2. CH 3 COO-NH4 +
Y
Example 116
General Description of Scheme I. A 1 dram vial is charged with either an aromatic or aliphatic
isocyanate in an inert solvent such as toluene. An equal or excess molar ratio of 2-amino-6-
nitrobenzothiazole or 2-amino-6-chlorobenzothiazole is added as a solid in one portion followed by
addition of an equal molar ratio of a base such as triethylamine. The reaction mixture is heated with
agitation in an incubator shaker at about 80 °C until the starting material is consumed. The
precipitated product is collected by standard methods and washed with ether.
Example 1
The following examples are representative of a synthesis in accordance with Scheme I.
Example 1A : A 1 dram vial was charged with 3,5-dimethoxyphenylisocyanate (51 mg, 0.282 mmol)
in 1 mL of toluene, and 2-amino-6-nitrobenzothiazole (50 mg, 0.256 mmol) was added as a solid in
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one portion followed by addition of triethylamine (36 |llL, 0.256 mmol). The reaction mixture was
heated with agitation in an incubator shaker at about 80 °C until the starting material was consumed.
The product precipitated and was collected on a fritted funnel and washed with diethyl ether. (M-H)
373, HPLC RT 2.99 min, *H NMR (5-DMSO) 3.76 (s, 3H), 3.75 (s, 3H), 6.25 (s, 1H), 6.74 ( s,
2H), 7.79 (d, 1H, J= 8), 8.2 (dd, 1H, J= 2 and J = 8), 8.98 (s, 1H), 9.19 (br s, 1H), 11.20 (br s, 1H).
Example IB: A 1 dram vial was charged with ethyl isocyanate (2.1 mL, 24.5 mmol), 2-amino-6-
chlorobenzothiazole (4.48 g, 24.3 mmol), and triethylamine (3.4 mL, 24.3 mmol) in 100 mL of
toluene. The reaction mixture was heated to reflux and the reaction progress monitored until the
starting material was consumed. The product was collected on a fritted funnel and washed with
diethyl ether to yield 5.68 g, (92%) of pure material. HPLC RT 1.96 min; (M-H)253; *H NMR (d-
DMSO) 5 1.10 (t, 3H), 3.2 (q, 2H), 6.72 (brs, 1H), 7.38 (d, 1H), 7.61 (d, 1H), 8.01 (s, 1H), 10.77 (br
s, 1H).
General Description of Scheme II. Synthesis of A of Scheme II. A round bottom flask is charged
with 2-amino~6-nitro-benzothiazoIe or 2-amino-6-chloro-benzothiazole and methyl
chlorothioformate in pyridine. The reaction mixture is heated at about 50 °C for about 8 hours and
cooled to room temperature overnight. The off-white solid is collected on a fritted funnel, washed
with diethyl ether, and dried in vacuo to yield the desired product.
A 1 dram vial is charged with A (1 eq) and the appropriate amine (about 1.2 eq or more) in
absolute EtOH. The reaction mixture is heated to about 80°C with agitation in an incubator shaker
until all of the starting material is consumed. Products that precipitate are collected on a fritted
funnel, washed with diethyl ether, and dried in vacuo. Products that do not precipitate are purified by
preparative reversed-phase HPLC.
Example 2
This example is representative of a synthesis in accordance with Scheme II. A 500 mL
round bottom flask was charged with 2-amino-6-nitro-benzathiazole (7.0 g, 0.036 mol) and methyl
chlorothioformate (6 g, 0.0543 mol) in 250 mL pyridine. The reaction mixture was heated at about
50 °C for about 8 hours and cooled to room temperature overnight. The off-white solid was collected
on a fritted funnel, washed with diethyl ether, and dried in vacuo to yield 4.6g, 47%, of product. (M-
H) 267.1, HPLC RT 3.22 min, lH NMR: (5-DMSO) 2.42 (s, 3H), 7.87 (d, 1H, J = 9), 8.27 (dd, 1H,
J = 2 and 9), 9.00 (s, 1 H), 1 3 .27 (br s, 1 H).
A 1 dram vial is charged with A (50 mg, 0.186 mmol) and 2-amino-2-methyl-propanol ( 20
mg, 0.223 mmol) in 1 mL of absolute ethanol. The reaction mixture was heated at about 80 °C for
about 14 hours or until the starting material was consumed. The product precipitated upon cooling
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and was collected on a fritted funnel, washed with diethyl ether and dried in vacuo. (M-H) 309.1;
HPLC RT 2.06 min; NMR (8-DMSO) 1.43 (s, 6H), 3.41 (d, 2H), 5.07 (t,lH), 6.67 (br s, 1H),
7.73 (d, 1H), 8.2 (d, 1H), 8.92 (s, 1H), 10.94 (br s, 1H).
General Description of Scheme III. Synthesis of B of Scheme ill. The synthesis of B is achieved
5 as described by Merchan et. al. Synthesis, 1982, 590. Recrystallization of the product is performed
using DMF.
A 1 dram vial is charged with B (1 eq) and the appropriate amine (about 1.2 eq) in absolute
EtOH. The reaction mixture is heated to about 80 °C with agitation in an incubator shaker until all
of the starting materials are consumed. Products that precipitate are collected on a fritted funnel,
10 washed with diethyl ether, and dried in vacuo. Products that do not precipitate are purified by
preparative HPLC.
Example 3
This example is representative of a synthesis jn accordance with Scheme 1IL To a stirring
solution of 2-amino-6-nitro-benzothiazole (7 g, 0.036 mol) in DMF at about 0 °C was added
15 dropwise NaOH (2.58 mL, 20M, 0.043 mol). The base was added in 3 portions with each addition
separated by about 20 min. A dark red color was observed. Carbon disulfide (4.33 mL, 0.072 mol)
was added dropwise over a period of about 10 minutes. The reaction mixture was stirred at about 0
°C for about 30 min. before another equivalent of NaOH was added in portions. Methyl iodide (2.23
mL, 0.036 mol) was added neat and the ice bath was removed. The reaction mixture was stirred at
20 room temperature for about 2 hours. The reaction mixture was poured into 200 mL of deionized
water and neutralized with 2 N HC1. The resulting suspension was stirred at room temperature
overnight and the precipitate collected on a fritted funnel. The product was isolated as long, yellow,
crystals. (M-H) 284; HPLC RT 2.69 min; 1"H NMR (S-DMSO) 2.86 (s, 3H), 7.71 (d, 1H), 8.3 (d,
1H), 8.98(s, 1H).
25 A 1 dram vial was charged with B (30 mg, 0.106 mmol) and ethylamine ( 63 uL (2M in
methanol), 0.126 mmol) in 1 mL of absolute ethanol. The reaction mixture was heated at about 80
°C for about 16 hours or until the starting material was consumed. The product precipitated upon
cooling and was collected on a fritted funnel, washed with diethyl ether and dried in vacuo. (M-H)
281; HPLC RT 2.74 min; *H NMR (S-DMSO) 1.1 (t, 3H), 3.5 (q, 2H), 7.7 (d, 1H), 8.2(d, 1H), 8.9
30 (s, 1H), 9.1 (brs, 1H), 12.15 (br s, 1H). (M-H) 284, HPLC RT 2.71 min, ] H NMR (8-DMSO) 2.61
(s, 3H), 7.72 (d, 1H, J - 11), 8.34 (dd, 1H, J = 2 and 9), 9.00 (d, 1H, J = 2).
General Description of Scheme IIIA. To a stirring suspension of 1A, formaldehyde and
methylamine in a solution of alcohol/water is added to N-methylmorpholine. The reaction mixture is
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heated to about 60 to 100 °C, preferably 80 °C, for about 18-20 hours. Reaction progress is
monitored by LCMS. The reaction is heterogeneous throughout the reaction. The desired product IB
is collected using standard methods in the art.
A round bottom flask is charged with IB and (phenylthio)acetonitrile in DMSO at room
5 temperature. A 1 M solution of potassium tert-butoxide in THF is added in one portion. The reaction
mixture is stirred at room temperature overnight. The crude reaction mixture is SLOWLY added to a
vigorously stirring mixture of ethyl acetate and ammonium acetate. The layers are separated and the
organic layer dried over anhydrous sodium sulfate and the desired product, 1C, isolated according to
standard methods known in the art.
10 To a stirring solution of 1C in DMSO is added a solution of potassium tert-butoxide (about 1
eq) in THF. Upon addition of the base the reaction mixture turned deep purple in color. An
equivalent of methyl iodide is added in one portion. The reaction mixture turned a deep red color.
The reaction mixture is stirred at room temperature for 1-6 hours. An aqueous solution of
ammonium acetate is added and the product extracted with methylene chloride. The crude product is
15 purified according to standard methods known in the art.
The triazone protecting group is removed under acidic conditions to yield the desired free
urea. Neat trifluoroacetic acid, 1 N aqueous hydrochloric acid, 4 M hydrochloric acid in dioxane and
a solution of 1:1 acetic acid in methanol all remove the protecting group at room temperature within
4-24 h. The preferred conditions for deprotection are 4 M HC1 in dioxane at room temperature until
20 the reaction is complete.
This example is representative of a synthesis in accordance with Scheme IIIA. To a stirring
suspension of 1A (555 mg, 2.09 mmol), formaldehyde (1.02 g, 20.8 mmol), and methylamine (354
25 |^L, 6.25 mmol) in a 1:1 (v/v) solution of ethanol/water was added N-methylmorpholine (583 jiL,
4.17 mmol). The reaction mixture was heated to about 80 °C for about 18-20 hours. Reaction
progress was monitored by LCMS. The reaction was heterogeneous throughout the reaction. The
solid was collected on a fritted funnel to yield 575 mg (86 %) of the desired product IB as yellow
Example 4
N
ci
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needles, mp 193-194 °C; LCMS MH + 321.9 m/z; ] H NMR (d-DMSO) 8 8.9 (1 H, s) 5 8.2 ( 1 H, d),
7.8(1 H,d), 5.1 (2H,s) 5 4.3(2H J s),3.3(2H,q),2.6(3H,s), 1.1 (3 H, t); 13 C NMR (d-DMSO) 8
164.6, 153,6, 151.0, 142.5, 133.5, 121.4, 120.0, 1 1 8.3, 69.1, 69.4, 38.5, 12.5.
A round bottom flask was charged with IB (500 mg, 1.56 mmol) and
5 (phenylthio)acetonitrile (279 jllL, 1.87 mmol) in 10 mL of DMSO at room temperature. A 1 M
solution of potassium tert-butoxide in THF (3.11 mL, 3.12 mmol) was added in one portion via
syringe. Upon addition of the base the reaction mixture turned deep purple in color. The reaction
mixture was stirred at room temperature overnight. The crude reaction mixture was SLOWLY added
to a vigorously stirring mixture of ethyl acetate and 100 mM ammonium acetate. The layers were
10 separated and the organic layer dried over anhydrous sodium sulfate. The solvent was removed in
vacuo to yield 29 1 mg (52%) of product as a light brown solid. LCMS: 3.02 min; MH+360 m/z.
To a stirring solution of 1C in DMSO was added a 1 M solution of potassium tert-butoxide
(1 eq) in THF. Upon addition of the base the reaction mixture turned deep purple in color. An
equivalent of methyl iodide was added in one portion. The reaction mixture turned a deep red color.
15 The reaction mixture was stirred at room temperature for 1-6 hours. An aqueous solution of
ammonium acetate (6M) was added and the product extracted with methylene chloride. The crude
product was purified by preparative HPLC/MS. MH + 375 m/z. The triazone protecting group was
removed under acidic conditions, as described hereinabove, to yield the desired free urea title
compound, LCMS: R.T. 2.66 min, MH-304 m/z.
20 Example 5
7V-(6-Chloro- 1 ,3-benzothiazol-2-yl>A' , -ethylurea
Three grams of 6-chloro-l,3-benzothiazol-2-amine was dissolved in about 50 mL DMF.
Next, about 2.5 mL of EtNCO was added followed by about 3.2 mL of triethylamine. The solution
was allowed to react at about 80 °C for about 8 hours. The reaction solvent was then removed in
25 vacuo and the crude oil was taken up in ether. The solids were isolated by filtration and washed with
ether. The product was then dried in vacuo. ^H NMR 1.09 (t, 3H, J - 7.2 Hz), 3.19 (m, 2H), 6.74 (br
s, 1H), 7.37 (d, 1H, J = 8.58 Hz), 7.60 (d, 1H, J = 8.59 Hz), 8.01 (s, 1H), 10.8 (br s, 1H). LCMS:
R.T. 2.3 min, MH-254 m/z.
Example 6
30 A / '-(6-Chloro-5-nitro-l,3-benzothiazol-2-yl)-A^'-ethylurea
Three grams of 7V-(6-chloro-l,3-benzothiazol-2-yl)-A^-ethylurea was dissolved in about 15
mL of concentrated sulfuric acid (about 92-94%). The solution was cooled to about 0-5°C. About
1 .5 a of ice cooled nitric acid (70% concentration was used, though this is not necessary) was added
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dropwise. The reaction was held at 0-5 °C for about one hour then poured into water. The pH was
then adjusted to about 7-8 with ammonia and the solids were isolated by filtration. The solids were
washed with water then dried in vacuo. The product was further purified by chromatography then
dried in vacuo. ^HNMR 1.08 (t, 3H, J = 7.2 Hz), 3.19 (m, 2H), 6.92 (br s, 1H), 8.26 (s, 1H), 8.31 (s,
5 1H), LC/MS 3.72 min, 302 (M+l)
Examples 7-166
10 The following Examples were synthesized substantially according to the example indicated
in the third column of the table using the appropriate starting material.
Example #
8
Structure
N
//
N
N
o
I.
N.
N
/
•N
O
Made
according
to example
1
1
Analytical
RP-HPLC
RT (min)
2.72
2.1
Mass Spec
(m/z)
329
267
N
N
N
1
2.68
293
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Example #
10
11
Structure
N.
Made v
according
to example
1
^1 o
N n
Analytical
RP-HPLC
RT (mln)
Mass Spec
(m/z)
2.41
2.73
279
397
12
2.37
337
13
14
' O.
1
A
/ — r N / c
2.67
2.94
379
373
15
1
3.57
449
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Example #
16
17
18
19
20
21
o
A:
■N,
Structure;
ci
•N
N
CI
N
N
Made
according
to example
"N
1
1
1
1
1
Analytical
RP-HPLC
RT (mm)
Mass Spec
(m/z)
2.78
2.83
2.81
2.27
2.5
2.84
381
327
347
277
293
365
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Example #
22
23
Structure
Made
according
to example
1
Analytical
RP-HPLC
RT (min)
Mass Spec
(m/z)
2.95
2.74
327
281
24
w/ ^
3.33
357
25
2.23
325
26
27
o'
I.
2.88
3.33
337
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Example #
Structure
Made ^
according
to example
Analytical
RP-HPLC
RT(min)
Mass Spec
(m/z) :
28
3.31
359
29
3.33
379 1
30
3.33
379^
31
2.89
333
32
1.79
366
33
2.09
299
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Example #
Structure
34
Made
r- i ^
according
to example
Analytical
RP-HPLC
RT (min)
Mass Spec
(m/z) ;
1.73
366
35
2.76
339
36
37
cr
1.6
1.76
350
308
38
39
6.54
5.08
292
278
84
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Example #
<jr - .
40
Structure
Made
according
to example
Analytical
RP-HPLC
RT(min)
5.26
Mass Spec
(m/z)
310
41
o
N
•N.
6.6
324
42
5.1
308
43
4
411
44
N
V
7.44
336
45
5.4
363
85
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Example #
Structure
.•• Made
according
to example
Analytical
RP-HPLC
RT(min)
Mass Spec
46
5.4
381
47
48
3.45
365
4.78
380
49
50
51
N N
N N
7.6
337
6.45
393
7.6
321
86
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87
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Example #
58
Structure
Made?
according
to example
Analytical
RP-HPLC
RT (min)
Mass Spec
(m/z)
2.92
339
59
2.07
350
60
3.16
422
61
2.9
372
62
2.64
343'
63
2.47
311
88
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Example #
64
65
66
67
68
69
o
i:
O
i:
Structure
Chiral
N-
"N 1,1
Chiral
Made
according
to example
Chiral
//
-N
Analytical
RP-HPLC
RT(min)
Mass Spec
\ (m/z)
2.59
2.59
2.39
2.11
2.08
2.7
311
311
311
327
327
371
89
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Example #
Structure
Made
according
to example
Analytical
RP-HPLC
RT(niln)
Mass Spec
{ (m/z)
70
o
i.
■N — " ^~-N
O
N
2.6
326
71
72
Chiral
3.16
2.91
353
353
73
3
N ^-N"
N
-N-
2.47
342'
74
75
N
N"^ N
nil O
2.31
2.4
341
389 1
90
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Example #
76
Structure
Made
according
to example
Analytical
RP-HPLC
RT (rnin)
Mass Spec
(m/z)
2.58
325
77
78
79
80
Chirat
Chiral
3.19
2.34
1.87
2.42
354
312
326
324
81
2.35
338
91
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Example #
Structure
Made
according
to example
Analytical
RP-HPLC
RT (min)
Mass Spec
: (rn/z)
82
1.56
325
83
o
L
■N.
N,
1.94
326
84
Chiral
2.18
310
85
2.42
343
86
Chiral
2.62
338
87
1.98
312
92
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Example #
88
Structure
Made
according
to example
Chirat
Analytical
RP-HPLC
RT (mln)
Mass Spec
(m/zy
2.04
296
89
Chiral
2.04
296
90
Chirat
2.01
296
91
1.96
296
92
NA
372
93
2.76
366
93
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Example*
94
95
o
i.
N.
Structure
N
, Made
according
to example
Analytical
RP-HPLC
RT (min)
Mass Spec
< (mlz) v
1.57
2.32
311
356
96
Chira!
2.19
355
97
o
L
^ — (
/
2.06
309
98
i.
N.
■N
2.94
321
99
3.67
363.8
94
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Example*
100
101
102
Structure
\ s Made ;
according
to example
Analytical
RP-HPLC
RT(min)
Mass Spec
}ss <m/z)
3.08
291
3.51
371
2.78
328
103
3.22
279
104
105
2.3
296
2.28
280
95
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Example #
Structure
; Wade
according
to example
Analytical
RP-HPLC
RT(min)
Mass Spec
(m/z)
106
3.54
355
107
2.21
308
108
3.6
363
109
o
I.
■ N,
N
Y
N-
3.19
421
110
3.6
363
111
o'
i:
//
•N'
3.53
96
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Example #
112
113
Structure
N
O
i:
O
•N
Made
according
to example
Analytical
RP-HPLC
RT(min)
Mass Spec
r (m/z)
2.64
3.2
306
375
114
o
-N
2.45
332
115
3.73
451
116
117
3A
CI
3.37
2.86
396
319
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Example*
118
119
120
121
122
123
Structure
Made
according
to example
o
o
w/ ^
■N
1
Analytical
RP-HPLC
RT (min)
Mass Spec
(m/z)
3.41
389
3.18
343
2.35
251
2.9
278
2.12
300
1.57
326
98
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Example #
124
Structure
Made ,
according
toexample
Analytical
RP-HPLC
RT (min)
Mass Spec
(m/z)
7.48
417
125
o"
I.
■ N.
V
6.72
400
126
8.35
429
127
128
129
8.96
413
8.32
320
8.19
439
99
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100
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Example*
Structure
136
137
v Made
according
to example
o
I
• N
//
N
Y'
•N
Analytical
RP-HPLC
RT(min)
Mass Spec
(m/z)
3.22
2.21
279
308
138
N
■N.
-N
3.12
361
139
2.7
283
140
L
N,
N
o
2.44
379
141
o
i.
• N,
O'
S
■N
N >
\
3.72
436
101
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Example #
Structure
Made
according
to example
Analytical
RP-HPLC
RT(min)
Mass Spec
(m/z)
142
N
II
2.13
326
143
1
2.26
2.23
370
370
144
145
1
1
2.88
2.92
282
316
146
1
3.84
396
147
1
3.27
334
102
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Example #
Structure
148
; Made
according
toexarnple
Analytical
RP-HPLC
:RT (mln)
Mass Spec
; ; (m/z) V ?
2.81
282
149
2.48
326
150
2.85
368
151
1
3.14
362
152
153
1
1
2.95
336
2.59
268
103
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104
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105
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Example*
j ?T :'* ' '' .-.■!-"'*' ■ ''i -■' ^i^' 5 - ' '•■ = : "' " ■■riji. f .;- - - !"' ^ .
■ /■ .,->;■.,.» r * "Viv^ ' : - ' • ' ;.. :
" " :-,v 'V; " % r - ' > . *■-'■■*■■ ■' = -r"" ■ v... -■ ".. ^ "■; --v'^ ^
; lj' , >■'■'' t ■ \* ' it:- y ; :l /^e. s / ]j.-?' v '(
Made ^
according
to example
Analytical
RP-HPLC.
RT(min)
Mass Spec
166
^ — N ^r[ /N
o
2
2.28
227
Note: NA = not available
The hydrogen(s) of hydroxy and amino groups are not shown in the above structural
formulas but instead are assumed to be present.
5
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Scheme IV
KSCN
Br 9) AcOH
NC
R 3 NCO
NC
NEt,, DMF
Pyridine,
MeSC(0)Cl
>
H
N
>
O
N — R'
H
NC
EtOH
NC
R 3 X 1 NH
3
/>
H
N
>
O
N— R'
^ 1
X 1
General Description of Scheme IV
5 2-Amino-l,3-benzothiazole-6-carbonitrile: 4-Aminobenzonitrile is dissolved in acetic acid (or a
weak protic acid) and the solution is cooled to about 16-30°C, preferably 16-18°C. Potassium
thiocyanate is added and the flask is then equipped with an addition funnel. The addition funnel is
charged with bromine and acetic acid. This dark solution is then added to the benzonitrile solution in
a dropwise fashion under good agitation and allowed to stir for about 12-20 hours, preferrably about
10 16 hours. The slurry is then drowned into water and filtered. The presscake is washed well with
water, reslurried in dilute aqueous alkali and filtered. Again the presscake is washed well with water
to obtain the title compound.
TV^e-Cyano-l^-benzothiazoW-yO-iV-ethylurea: 2-Amino- 1 ,3-benzothiazoIe-6-carbonitrile is
dissolved in a polar aprotic solvent, preferrably dimethylformamide. R 3 NCO is added, followed by
15 an alkyl amine base, preferably triethylamine and the solution is heated to about 70-90°C, preferably
about 80°C, under good agitation. The solution is allowed to stir for about 4-8 hours, preferably
about 4 hours, then cooled to room temperature. The solvent is removed in vacuo and the solids are
washed well with ether. The product is further purified by column chromatography and after drying
in vacuo, the title product is isolated.
20 Methyl [(6-cyano-l,3-benzothiazol-2-yl)amino]methanethioate: 2-Amino-l,3-benzothiazole-6-
carbonitrile is dissolved in pyridine. Methyl chlorothiolformate is added and the solution is heated to
about 50-60°C, preferably about 50°C, under good agitation. The solution is allowed to stir for about
8-24 hours, preferably 8 hours, then cooled to room temperature. The slurry is filtered and the solids
are washed well with water and dried in vacuo.
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Synthesis of Compound C of Scheme IV: Methyl [(6-cyano-l,3-benzothiazol-2-
yl)amino]methanethioate is dissolved in alkanol. An excess of the appropriate amine, R 3 X ] NH is
added, and the solution is heated to about 75-85°C, preferably 80°C ? under good agitation. The
solution is allowed to stir for about 8-24 hours, preferably 14 hours, then cooled to room
5 temperature. The solvent is removed in vacuo. The product can be further purified by column
chromatography to yield the desired product.
Preparation 1
2-Amino-l,3-benzothiazole-6-carbonitrile
Two grams of 4-aminobenzonitrile was dissolved in about 40 mL acetic acid and the
10 solution was cooled to about 16°C. About 3.3g of potassium thiocyanate was added and the flask
was equipped with an addition funnel. The addition funnel was charged with about 2.7 g bromine
and about 5 mL acetic acid. This dark solution was then added to the benzonitrile solution in a
dropwise fashion under good agitation and allowed to stir for about 16 hours. The slurry was then
drowned into water and filtered. The presscake was washed well with water, reslurried in dilute
15 aqueous alkali and filtered. Again the presscake was washed well with water. After drying in vacuo,
about 2 grams of the title compound was isolated. J H NMR 6.8 (d, 1H, J = 8.7 Hz), 6.9 (br s, 2H),
7.6 (dd, 1H, J = 2 Hz, J = 8.7 Hz), 8.0 (d, 1H, J = 2 Hz), LC/MS 2.34 min, 174 (M-H-), RP-HPLC
RT 7.7 minutes.
Preparation 2
20 Methyl [(6-cyano-l,3-benzothiazol-2-yl)amino]methanethioate
2-Amino-l,3-benzothiazole-6-carbonitrile (1.4 g) was dissolved in about 50 mL pyridine.
About 2 g of methylchlorothiolformate was added and the solution was heated to about 50°C under
good agitation. The solution was allowed to stir for about 8 hours then cooled to room temperature.
The slurry was filtered and the solids were washed well with water. After drying in vacuo, about 1 .2
25 grams of the title product was isolated. ^H NMR 2.4 (s, 3H), 7.8 (m, 2H), 8.5 (s, 1H), 13.2 (br s,
1 H), LC/MS 2.92 min, 250 (MH+), 248 (M-H~).
Instrumentation for Examples 166-197 were:
LC/MS purification conditions:
Column: Hypersil®BDS, CI 8, 5ja, 100x21.2 mm (Hypersil Inc., Needham, MA)
30 Gradient: Generally from 100% pH 4.5 50mM NH4OAC/H2O to 100% CH3CN in 8.5
minutes but varies depending upon required separation
Flow rate: 25 mL/min
lH NMR spectrum
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Recorded on a Bruker 400 MHz spectrometer in deuterated DMSO using tetramethylsilane (0.00
ppm) as internal standard.
LC conditions (analytical run):
Column: PECOSPHERE, CI 8, 3jnm, 33x4.6mm (Perkin Elmer, Norwalk, CT)
5 Gradient: From 1 00% pH 4.5 50mM NH4OAC/H2O to 1 00% CH3CN in 4.5 minutes
Flow rate: 3.5 mL/min
Example 167-169
A^-Cyano-l^-benzothiazol-Z-yO-A^-ethylurea
2-Amino-l,3-benzothiazole-6-carbonitrile (0.2 g) was dissolved in about 5 mL
10 dimethylformamide. About 0.2 mL of ethyl isocyanate was added, followed by about 0.3 mL
triethylamine and the solution was heated to about 80°C under good agitation. The solution was
allowed to stir for about 4 hours then cooled to room temperature. The solvent was removed in
vacuo and the solids were washed well with ether. The product was further purified by column
chromatography and after drying in vacuo, about 0.14 grams was isolated. lH NMR 1.1 (t, 3H, J =
15 7.2 Hz), 3.2 (m, 2H), 6.8 (s, 1H), 7.7 (m, 2H), 8.4 (s, 1H), 1 1.0 (s, 1H), LC/MS 2.54 min, 247
(MH+), 245 (M-H-), lab RP-HPLC RT 7.8 minutes.
Examples 168 and 169 were synthesized according to the synthesis of Example 167 using
the appropriate starting material.
Example 1 68: 7V-(6-Cyano-l^-benzothiazol-2-yl)-7V-[(15^-l-phenylethyl]urea: ^ NMR 1.4 (d,
20 3H, J = 6.8 Hz), 4.9 (m, 1H), 7.3 (m, 6H), 7.7 (br s, 2H), 8.4 (s, 1H), 10.8 (br s, 1H), LC/MS 3.32
min, 323 (MH + ), 321 (M-H-).
Example 1 69: ^-(e-Cyano-l^-benzothiazol^-yQ-TV^-Kljgj-l-phenylethyllurea: *H NMR 1.4 (d,
3H, J = 6.9 Hz), 4.9 (m, 1H), 7.3 (m, 5H), 7.36 (d, 1H, J = 4.5 Hz), 7.7 (m, 2H), 8.4 (s, 1H), 10.8 (br
s, 1H), LC/MS 3.30 min, 321 (M-H").
25 Examples 170-171
The following examples were synthesized in accordance with the general procedure for
making a compound C of Scheme IV, using the appropriate amine.
Example 1 70: 7Y-(6-Cyano-l^-benzothiazol-2-yl)-A^-(4-pyridylmethyl)urea: ] H NMR 4.4 (d, 2H,
J = 6 Hz), 7.3 1 (d, 2H, J = 5.8 Hz), 7.4 (br s, 1H), 7.8 (m, 2H), 8.46 (s, 1H), 8.52 (d, 2H, J = 5.8 Hz),
30 1 1.3(br s, 1H), LC/MS 2.44 min 310 (MH+), 308 (M-H").
Example 171: 7Y-(6-Cyano-l^-benzothiazol-2-yl)-/V-(3-pyridylmethyl)urea: *H NMR 4.4 (d, 2H,
J = 5.9 Hz), 7.38 (m, 2H), 7.8 (m, 3H), 8.46 (m, 2H), 8.55 (s, 1H), 11.1 (br s, 1H), LC/MS 2.46 min,
310 (MH+), 308 (M-H").
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Scheme V
General procedure for synthesizing l-ethyl-5-methyl-3-(6-nitro-l,3-benzothiazol-2-yI)-l^,5-
triazinan-2-one: A^-Ethyl-A^-(6-nitro-l,3-benzothiazol-2-yl)urea is suspended in an alkanol/water
mixture, preferably 1:1 EtOH/H20 at room temperature. A 37% aqueous solution of formaldehyde
is added followed by addition of a 2M solution of MeNH2 in MeOH, then about 2 mole equivalents
of N-methylmorpholine. The solution is warmed to about 70~85°C, preferably 80°C, then allowed to
stir for about 4-24 hours, preferably 4 hours. The slurry is then cooled to room temperature, filtered
and washed well with water, dried and the title product is isolated.
General procedure for Grignard addition:
The Grignard synthesis can be performed substantially according to the method of Bartoli,
JOC, (1980), 45, 522-524. For example, l-ethyl-5-methyl-3-(6-nitro-l,3-benzothiazoI-2-yI> 1,3,5-
triazinan-2-one is dissolved in an inert solvent such as an ether, preferably tetrahydrofuran. This
slurry is cooled to about 0-5°C, preferably 0°C. About 2 molar equivalents of the appropriate
Grignard reagent is added dropwise to the slurry. Once addition is complete the solution is stirred at
about Q-30°C for about 5 minutes. Next, about 0.66 mole equivalents of KMnC>4, dissolved in 1:1
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acetone/water, is added dropwise at about 0-5°C, preferably 0°C. The solution is allowed to stir to
room temperature. The reaction crude is then diluted with water and the desired product is extracted
into methylene chloride. The combined organic layers are dried over magnesium sulfate, then the
solvent is removed in vacuo. The product can be further purified by chromatography then dried in
5 vacuo,
Preparation 3
l-EthyI-5-methyl-3-(6-nitro-l,3-benzothiazol-2-yI)-l,3,5-triazinan-2-one
A^Ethyl-JV-(6-nitro-U3-benzothiazol-2-yl)urea (2.8 g) was suspended in about 100 mL 1:1
EtOH/UbO at room temperature. About 8 mL of a 37% aqueous solution of formaldehyde is added
10 followed by addition of about 15 mL of a 2M solution of MeNH2 in MeOH, then about 2.2 mL of
N-methylmorpholine. The solution was warmed to about 80°C then allowed to stir for about 16
hours. The slurry was then cooled to room temperature, filtered and washed well with water. After
drying in vacuo, about 3.2 grams was isolated. ] H NMR 1.13 (t, 3H, J = 7.1 Hz), 2.55 (s, 3H), 3.38
(q, 2H, J = 7.1 Hz), 4.39 (s, 2H), 5.16 (s, 2H), 7.81 (d, 1H, J = 8.9 Hz), 8.22 (dd, 1H, J - 2.4 Hz, J =
15 8.9 Hz), 8.95 (d, 1H, J -2.4 Hz) LC/MS = 3.33 min, 322 (MH + ), 320 (M-H~).
Examples 1 72 — 1 77
The following examples were synthesized according to the foregoing synthetic description
using the noted Grignard reagent.
Example 172 : l-EthyI-3-(7-ethyl-6-nitro-13^
20 Grignard reagent - EtMgBr in Et20; *H NMR 1.13 (t, 3 H, J = 7.1 Hz), 1.33 (t, 3H, J = 7.5 Hz),
2.55 (s, 3H), 3.05 (m, 2H), 3.35 (m, 2H), 4.4 (s, 2H), 5.15 (s, 2H) S 7.68 (d, 1H, J - 8.84 Hz), 8.02 (d,
1H, J = 8.83 Hz), LC/MS 3.61 min, 351 (MH + ), 349 (M-H").
Example 173: l-(7-AlIyl-6-nitro-l,3-benzothiazoI-2-yl)-3-ethyl-5-methyl-l^,5-triazinan-2-one
Grignard reagent = Allyl-MgBr in THF; lH NMR 1.12 (t, 3H, J - 7.1 Hz), 2.54 (s, 3H), 3.38 (m,
25 2H), 3.82 (d, 2H, J = 6.2 Hz), 4.4 (s, 2H), 5.02 (s, 1H), 5.06 (d, 1H, J = 1 .6 Hz), 5.1 (s, 2H), 6.02 (m,
2H), 7.72 (d, 1H, J = 8.8 Hz), 8.06 (d, 1H, J = 8.8 Hz), LC/MS 3.6 min, 362 (MH+), 361 (M-H-).
Example 1 74: l-Ethyl-5-methyl-3-(6-nitro-7-phenyl-l,3-benzothiazol-2-yl)-l^,5-triazinan-2-on^
Grignard reagent -Phenyl-MgCl in THF; LC/MS 2.77 min., 398 (MH + ).
Example 175 : l-(7-benzyl-6-nitro-l^-benzotta^
30 Grignard reagent = Benzyl-MgCl in THF; 'H NMR 1.09 (t, 3H, J - 7.1 Hz), 2.52 (s, ( 3H). 3.3 (m,
2H), 4.36 (s, 2H), 4.49 (s, 2H), 5.12 (s, 2H) 5 7.12 (m, 2H), 7.19 (m, 1H), 7.28 (m, 2H), 7.76 (d, 1H,
J = 8.8 Hz), 8.09 (d, 1H, J - 8.8 Hz), LC/MS 3.81 min, 412 (MH+).
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Example 1 76 : l-Ethyl-3-(7-methy]-6-nitro-l^-benzothiazoI-2-yl)-5-methyl-l^,5-triazinan-2-
one
Grignard reagent - MeMgCl in THF; NMR 1.13 (m, 3H), 2.54 (s, 3H), 2.73 (s, 3H), 3J5 (m,
2H), 4.39 (s, 2H), 5.15 (s, 2H), 7.61 (d, 1H, J = 8.4 Hz), 8.05 (d, 1H, J = 8.8 Hz), LC/MS 336 min,
5 336 (MH + X 335 (M-H").
Example 177: tert-Buty\ 2-(2-(3-ethyI-5-methyl-2-oxo-l^,5-triazinan"l-yI)-6-nitro-13-
benzothiazol-7-yl)-acetate
1 -Ethyl-5-methy l-3-(6-nitro- 1 ,3^benzothiazol-2-yl)- 1 ,3 ,5-triazinan-2-one (0.05 g) was
dissolved in about 25 mL dimethylformamide. The solution was cooled to about -40°C. About 0.24
10 mL of ter/-butyIchloroacetate was added dropwise. Next, about 1 .5 mL of KOr-Bu in THF (1M) was
introduced in a dropwise fashion. Once complete the solution was stirred at about -40 to -50°C for
about 3 hours. Next, about 2 mL of saturated ammonium chloride was added and the solution was
warmed to room temperature. The reaction crude was then diluted with water and the product was
extracted into ethyl acetate. The combined organic layers were dried over magnesium sulfate, then
15 the solvent was removed in vacuo. The product was further purified by chromatography then dried
in vacuo. 1 H NMR I.I (t, 3H, J = 7.0 Hz), 1-23 (s, 9H), 2.55 (s, 3H), 3.38 (m, 2H), 4.1 (s, 2H), 4.4
(s, 2H), 5.16 (s, 2H), 7.7 (d, 1H, J = 8.8 Hz), 8.1 (d, 1H, J = 8.8 Hz), LC/MS 3.72 min, 436 (MH+).
General procedure for hydrolysis of urea protecting group
The appropriate 7-substituted 1 -ethyl-5-methyl-3-(6-nitro- 1 ? 3-benzothiazol-2-yl)- 1 ,3,5-
20 triazinan-2-one is dissolved in excess protic acid (such as trifluoroacetic acid or aqueous HC1) and
stirred at room temperature until complete. Following neutralization, the products are either filtered
and washed with water or extracted into methylene chloride then dried. Products are further purified
by chromatography. The products are dried in vacuo.
Examples 178-183
25 The following examples were synthesized according the foregoing general description for
hydrolysis.
Example 178 : fer/-ButyI 2-(2-[(ethylamino)carbonyI]amino-6-nitro-l,3-benzothiazol-7-
yl)acetate
^H NMR 1.1 (t,3H, J -7.2 Hz), 1.4 (s, 9H) 5 3.2 (m, 2H), 4.1 (s, 2H), 6.9 (br s, 1H), 7.69 (d, 1H, J«
30 8.8 Hz), 8.14 (d, 1H, J = 8.8 Hz), 11.25(brs, 1H), LC/MS 3.32 min, 381 (MH+).
Example 1 79 : A^Ethyl^-CT-ethyl-e-nitro-lJ-benzothiazol^-vI^urea
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lH NMR, 1.1 (t, 3H, J = 7.2 Hz), 1.3 (t, 3H, J = 7.4 Hz), 3.0 (m, 2H), 3.2 (m, 2H), 7.0 (br s, 1H),
7.59 (d, 1H, J = 8.8 Hz), 8.01 (d, 1H, J = 8.8 Hz), 1 1.35 (br s, 1H), LC/MS 3.13 min., 295 (MH+),
293 (M-H-).
Example 1 80 : A47-Allyl-6-nitro-l,3-benzothiazol-2-yl)-A^-ethylurea
lH NMR 1.1 (t, 3H, J = 7.2 Hz), 3.2 (m, 2H), 3.82 (d, 2H, J = 6 Hz), 5.06 (d, 1H, J = 1.6 Hz), 5.12
(d, 1H, J = 9.3 Hz), 6.0 (m, 1H), 6.8 (br s, 1H), 7.65 (d, 1H, J = 8.8 Hz), 8.06 (d, 1H, J = 8.8 Hz),
1 1 .17 (br s, 1H), LC/MS 3.04 min., 307 (MH+), 305 (M-H").
Example 181 : Af-(7-Benzyl-6-nitro-l^-benzothiazol-2-yl)-iV-ethylurea
lH NMR 1.07 (t, 3H, J = 7.2 Hz), 3.15 (m, 2H), 4.49 (s, 2H), 6.77 (br s, 1H), 7.11-7.29 (m, 5H),
7.69 (d, 1H, J = 8.4 Hz), 8.09 (d, 1H, J = 8.8 Hz), 1 1.15 (br s, 1H), LC/MS 3.44 min, 357 (MH+),
355 (M-H").
Example 182 : A^Ethyl-A^-^-nitro-V-phenyl-l^-benzothiazoW-yOurea
LC/MS 2.51 min., 341 (MH+), 343 (M-H").
Example 183 : 7Y-Ethyl-A''-(7-inethyl-6-nitro-l,3-benzothiazol-2-yl)urea
1 H NMR, 1.1 (t, 3H, J = 7.1 Hz), 2.73 (s, 3H), 3.2 (m, 2H), 6.79 (br s, 1H), 7.61 (d, 1H, J = 8.8 Hz),
8.05 (d, 1H, J = 8.8 Hz), 1 1.15 (br s, 1H), LC/MS 2.85 min., 281 (MH+), 279 (M-H").
Scheme VI
O
KBrOs, HBr
H 2 0, rt
Thiourea
THF, rt, 2 days
O
S-^NH
O
2
NH.
Dioxane,
Reflux
O
NH
O
CH^CH ? NCO y
2 Et,N, DMF, 90°C
O
A
N N
H H
Preparation 4
2-Bromo-l, 3-cyclohexadione (1). To a solution of 1, 3-cyclohexadione (1.15 g, 10.0 mol, 97%
pure) and 48% HBr (aq) (1.5 mL, 13.3 mmol, 1.33 eq) in H2O (10 mL) at about 20°C, a warm
solution of KBr03 (0.55 g, 3.30 mol, 0.33 eq) in H2O (10 mL) was added dropwise over about 10
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min. It was important to keep the solution of KBr03/H20 around 35°C in order to keep the
potassium salt dissolved. The reaction mixture turned warm upon addition and was stirred at about
20°C for about 15 min. The precipitation was filtered off and washed with H2O (3x5 mL). The
solid was dried under vacuum to give 1.68 g (88%) of 1. The material was used in the following
5 synthesis without further purification. ] H NMR (CDCI3) 8 6.52 (br s, 1H), 2.62 (m 5 4H, CH 2 \ 2.03
(p, 2H, J = 6.4 Hz, CH 2 y
Preparation 5
2-Amino-7-oxo-4, 5, 6, 7-tetrahydro-benzothiazole (3). A suspension of .2-bromo-l,3-
cyclohexadione 1 (15.44 g, 80.8 mmol) and thiourea (6.15 g, 80.8 mmol, 1.0 eq) in anhydrous THF
10 (120 mL) was stirred at about 20°C for about 2 days. The disappearance of 1 and the appearance of
2 could be seen on TLC. The mixture was concentrated and anhydrous dioxane (120 mL) was added.
The reaction mixture was heated at about 110°C for about 1 day. It was cooled down and the
precipitation was filtered off and washed with THF (2 x 150 mL). The solid was dissolved in H2O
(100 mL) and neutralized with sat. NaHCC>3 solution whereupon a precipitate formed. The
15 precipitate was collected and recrystallized from MeOH to give 7.93 g (58%) of 3. *H NMR
(DMSO) 5 8.10 (br s, 2H, N/7?), 2.67 (t, 2H, J - 6.0 Hz, CH 2 \ 2.36 (t, 2H, J - 6.0 Hz, CH 2 % 1.99
(p, 2H, J = 6.4 Hz, CH 2 ); Mp 259.3-262.5°C (Decomposed).
Preparation 6
l"(7"Oxo-4,5,6 ? 7-tetrahydro-2-benzothiazolyI)-3-ethyl-urea (4). A solution of 2-amino-7-oxo-
20 4,5,6,7-tetrahydro-benzothiazole 3 (11.56 g, 68.7 mmol) in anhydrous DMF (200 mL) was treated
with triethylamine (19.2 mL, 137 mmol, 2.0 eq) and ethyl isocyanate (10.9 mL, 137 mmol, 2.0 eq).
The reaction mixture was heated at about 90°C with stirring for about 3 hours. The DMF solvent
was distilled off under reduced pressure. A sticky brown residue was obtained. Treatment with Et 2 0
(100 mL) gave a precipitate, which was filtered off and washed with more Et20 (50 mL). The light
25 brown colored solid was dried under vacuum to give 13.97 g (85%) of 4. The material was used in
the following synthesis without further purification. ] HNMR (DMSO) 8 10.95 (br s, 1H, NH), 6.66
(br s, 1H, N#), 3.16 (p, 2H, J = 7.2 Hz, CH 2 \ 2.79 (t, 2H, J = 6.1 Hz, CH 2 \ 2.45 (t, 2H, J = 6.5 Hz,
CH?X 2.05 (p, 2H, J = 6.4 Hz, CH 2 ), 1.07 (t, 3H, J = 7.2 Hz, CH3); LC/MS 240 (MH + ); RP-HPLC
RT 2.27 minutes.
30 Scheme VII
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o
o
A
N N
H H
Br 9) HBr
HOAc, 45°C
DBU
THF, rt
Preparation 7
l-(6,6-Dibromo-7-oxo-4,5,6,7-tetrahydro-2-benzothiazoIyl)-3-ethyI-urea (5 ). A solution of l-(7-
oxo-4 5 5,6,7-tetrahydro-2-benzothiazolyl)-3-ethyl-urea 4 (5.00 g, 20.9 mmol), 48% HBr (aq) (1.20
5 mL, 2.09 mmol, 0.1 eq) and AcOH (45 mL) was treated dropwise with a solution of Br2 (2.21 mL,
42.8 mmol, 2.05 eq) in AcOH (5 mL) with stirring. The reaction mixture was heated at about 45°C
with stirring for about 16 hours with a condensor on top of the reaction flask. An orange colored
suspension was obtained. The solid was filtered off and washed with Et20 (20 mL), toluene (50 mL)
and Et20 (3 x 30 mL). After drying under vacuum, 7.78 g (94%) of compound 5 was obtained. ! H
10 NMR (DMSO) 5 1 1 .35 (br s, 1H, KH), 6.78 (br s, 1H, N#), 3.18 (m, 2H, CH 2 \ 3.12 (t, 2H, J = 5.6
Hz, CH 2 \ 2.91 (t, 2H, J = 5.6 Hz, CH 2 \ 1-08 (t, 3H, J = 7.2 Hz, Ctfj); LC/MS 396 (MH + ); RP-
HPLC RT 3.04 minutes.
Example 184
l-(6-Bromo-7-hydroxy-2-benzothiazoIyl)-3-ethyl-urea (6). A suspension of l-(6,6~dibromo-7-
15 oxo-4,5,6,7-tetrahydro-2-benzothiazolyl)-3-ethyl-urea 5 (7.78 g, 19.6 mmol) in THF (50 mL) was
treated with DBU (8.79 mL, 58.8 mmol, 3.0 eq) dropwise at about 20°C. A dark green suspension
was obtained while heat was generated upon addition of DBU. It was stirred at about 20°C for about
18 hours. The reaction mixture was concentrated and the residue was treated with saturated NH4CI
(aq) solution to neutral. A light brown colored precipitate was obtained. It was filtered off and
20 washed with H2O (2 x 50 mL), small amount of MeOH and CH2C12, and finally dried under
vacuum to give 4.83 g (78%) of the desired compound 6. ] HNMR (DMSO) 8 10.68 (br s, 1H, N#),
7.43 (d, 1H, J = 8.5 Hz, ArH), 7.08 (d, 1H, J - 8.5 Hz, Ar#), 6.70 (br s, 1H, Nif), 3.18 (m, 2H,
CH 2 \ 1-09 (t, 3H, J = 7.2 Hz, CH3). LC/MS 316 (MH+); RP-HPLC RT 2.80 minutes.
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Scheme VIII
o
//
o
A
N N
H H
Br„ HBr
HOAc, 45°C
DBU
THF, rt
OH
8
O
N
H
N
H
Preparation 8
5 l-(6-Bromo-7-oxo-4,5,6,7"tetrahydro-2-benzothiazolyl)-3-ethyl"Urea (7). A solution of l-(7-
oxo-4,5 s 6,7-tetrahydro-2-benzothiazolyl)-3-ethyl-urea 4 (1.00 g, 4.18 mmol) and 48% HBr (aq)
(0.24 mL, 2.09 mmol, 0.5 eq) in AcOH (18 mL) was treated dropwise with a solution of Br2 (0.23
mL, 4.39 mmol, 1.05 eq) in AcOH (1 mL) with stirring. The reaction mixture was heated at about
45°C with stirring for about 16 hours with a condensor on top of the reaction flask. An orange
10 colored suspension was obtained. The solid was filtered off and washed with AcOH (5 mL), toluene
(2x3 mL) and Et20 (2x5 mL). After drying under vacuum, 1.1 1 g (83%) of the desired compound
7 was obtained. 1 H NMR (DMSO) 5 1 1 . 1 5 (br s, 1 H, NJ7), 6.73 (br s, 1 H, NH), 4.87 (t, 1 H, J = 4.6
Hz, CH), 3.17 (m, 2H, CH 2 \ 2.87 (dd, 2H, J = 7.2, 4.4 Hz, CH 2 ), 2.61-2.54 (m, 1H, CH 2 \ 2.39-
2.33 (m, 1H, CHi), 1.08 (t, 3H, J - 7.2 Hz, CH3); LC/MS 318 (MH + ); RP-HPLC RT2.68 minutes.
15 Preparation 9
l-(7-Hydroxy-2-benzothiazolyI)-3-ethyl-urea (8). To a suspension of l-(6-bromo-7-oxo~4,5,6,7-
tetrahydro-2-benzothiazolyl)-3-ethyl-urea 5 (0.100 g, 0.314 mmol) in THF (1.0 mL) was added
DBU (0.141 mL, 0.94 mmol, 3 eq) dropwise at about 20°C. A dark green suspension was obtained
while heat was generated upon addition of DBU. It was stirred at about 20°C for about 1 8 hours. The
20 reaction mixture was concentrated and dissolved in DMF (2 mL). LC/MS purification gave 0.024 g
(32%) of the desired compound 8. ^H NMR (DMSO) 5 10.54 (br s, 1H, N#), 7.17-7.07 (m, 2H,
Axfl), 6.63 (d, 1H, J = 6.9 Hz, ArJT), 6.70 (br s, 1 H, NH), 3.18 (m, 2H, CH 2 \ 1.09 (t, 3H, J = 7.2 Hz,
CH3). LC/MS 238 (MH + ); RP-HPLC RT 2.26 minutes.
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5 Example 185
Example 186
Example 187
Example 188
Example 189
10 Example 190
Example 191
Compound
9
10
11
12
13
14
15
Rl
C 6 H 5 CH 2 -
CH 3 -
(CH 3 ) 2 CH-
CH 3 0(CH 2 )20(CH 2 )2-
p-F-C 6 H 4 CH 2 -
CF 3 S0 2 -
NH 2 COC(CH 3 ) 2 -
General Procedure for l-(6-bromo-7-aIkoxy-2-benzothiazolyl)-3-ethyl-urea compounds 9-13. A
mixture of l-(6-bromo-7-hydroxy-2-benzothiazolyl)-3-ethyl-urea 6 and potassium carbonate (1.05
15 eq) in anhydrous DMF was stirred at about 20°C for about 0.5 hour, and was cooled down to about
0°C. The mixture was treated with an alky! halide (1.0 eq), and was stirred at about 0 - 85°C for
about 16 hours. To the reaction mixture was added methanol. The solid was filtered off and washed
with methanol. The solvent was evaporated and the residue was dissolved in DMF. The crude
reaction solution was purified by preparature LC/MS to give pure desired product:
20 Example 185
1- (6-Bromo-7-benzyloxy-2-benzothiazolyl)-3-ethyl-urea (9). A mixture of l-(6-bromo-7-hydroxy-
2- benzothiazolyl)-3-ethyl-urea 6 (0.050 g, 0.16 mmol) and potassium carbonate (0.023 g, 0.17
mmol, 1.05 eq) in anhydrous DMF (1.6 mL) was stirred at about 20°C for about 0.5 hour, and was
cooled down to about 0°C. The reaction mixture was treated with benzyl bromide (0.019 mL, 0.16
25 mmol, 1.0 eq), and was stirred at about 0°C for about 16 hours. To the reaction mixture was added
methanol (10 mL). The solid was filtered off and rinsed with methanol (3 mL). The solvent was
evaporated and the residue was dissolved in DMF (2 mL). The crude reaction solution was purified
by LC/MS to give 0.029 g (45%) of the desired compound 9. ] H NMR (DMSO) 5 10.86 (br s, 1H,
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N#), 7.59-7.34 (m, 7H, ArH) 9 6.72 (br s, 1H, N#), 5.17 (s, 2H, CH 2 \ 3.18 (m, 2H, CH 2 \ 1-09 (t,
3H, J = 7.2 Hz, CH 3 )\ LC/MS 406 (MH + ); RP-HPLC RT 3.8 minutes.
Example 1 86
l-(6-Bromo-7-methoxy-2-benzothiazolyl)-3-ethyl-urea (10). A mixture of l-(6-bromo-7-hydroxy-
5 2-benzothiazolyl)-3-ethyl-urea 6, potassium carbonate and iodomethane in DMF was reacted to give
0.0034 g (2%) of the desired compound 10. 'HNMR (DMSO) 8 10.88 (br s, 1H, N/Z), 7.55 (d, 1H,
J = 8.5 Hz, Ar#), 7.33 (d, 1H, J - 8.5 Hz, ArJZ), 6.82 (br s, 1H, NiZ), 3.93 (s, 3H, CH 3 \ 3.18 (m,
2H, CH 2 \ 1 .09 (t, 3H, J - 7.2 Hz, CH 3 ); LC/MS 330 (MH+); RP-HPLC RT 3.04 minutes.
Example 187
10 l-(6-Bromo-7-isopropoxy-2-benzothiazolyl)-3-ethyl-urea (11). A mixture of l-(6-bromo-7-
hydroxy-2-benzothiazolyl)-3-ethyl-urea 6, potassium carbonate and 2-bromopropane in DMF was
reacted to give 0.046 g (81%) of the desired compound 11. ] H NMR (DMSO) 5 10.83 (br s, 1H,
KH), 7.55 (d, 1H, J = 8.5 Hz, ArH) 9 7.31 (d, 1H, J = 8.6 Hz, ArH), 6.71 (br s, 1H, NH), 4.69 (hept,
1H, J = 6.0 Hz, CH), 3.18 (m, 2H, CH 2 ), 1.32 (d, 6H, J = 6.1 Hz, CH 3 ),\$9 (t, 3H, J - 7.2 Hz,
15 CH 3 ); LC/MS 358 (MH+); RP-HPLC RT 3.42 minutes.
Example 1 88
l-(6-Bromo-7-(2-(2-methoxyethoxy)ethoxy)-2-benzothiazolyI)-3-ethyl-urea (12). A mixture of 1-
(6-bromo-7-hydroxy-2-benzothiazolyl)-3-ethyl-urea 6, potassium carbonate and 2-(2-
methoxyethoxy)ethyl bromide in DMF was reacted to give 0.026 g (39%) of the desired compound
20 12. ^H NMR (DMSO) 5 10.65 (br s, 1H, N/f), 7.54 (d, 1H, J = 8.5 Hz, ArH), 7.32 (d, 1H, J - 8.6
Hz, ArH), 6.73 (br s, 1H, NTT), 4.24 (d, 2H, J - 4.4 Hz, CH 2 ), 3.76 (d, 2H, J = 4.8 Hz, CH 2 ), 3.61
(dd, 2H, J = 6.0, 5.2 Hz, CH 2 ), 3.48 (dd, 2H, J = 6.0, 5.2 Hz, CH 2 ), 3.26 (s, 3H CH 3 ), 3.19 (m, 2H,
CH 2 ), 1 .09 (t, 3H, J = 7.1 Hz, CH 3 ); LC/MS 418 (MH + ); RP-HPLC RT 2.95 minutes.
Example 189
25 l-(6-Bromo-7-(4-fluoro-benzyloxy)-2-benzothiazoIyI)-3-ethyI-urea, (13): A mixture of 1(6-
bromo-7-hydroxy-2-benzothiazolyl)-3-ethyl-urea 6, potassium carbonate and p-fluoro-benzyl-
bromide in DMF was reacted to give 0.013 g (19%) of the desired compound 13. ^H NMR (DMSO)
5 1.08 (t, 3H, J - 8Hz CH 2 C/fj), 3.18 (m, 2H, CH 2 ), 5.16 (s, 2H, OCH 2 Ai), 6.73 (br s, 1H, N#),
7.26 (dd, 2H, J - 4, 4 Hz, ArH) J 36 (d, 1H, J - 8 Hz, ArH), 7.56 (d, 1H, J = 4 Hz, ArH), 7.58 (d,
30 2H, J = 4 Hz, ArH), 10.89 (br s, 1H, N//). HPLC retention time 3.61 minutes.
Example 190
l-(6-Bromo-7-trifluoromethanesuIfonyl-2-benzthiazolyl)-3-ethyI-urea y (14): To a solution of 1-
(6-bromo-7-hydroxy-2-benzothiazolyl)-3-ethyl-urea (50 mg, 0.158 mmol) in pyridine (1 mL) were
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added 3 portions of (CF3SC>2)20 (29 pL, 0.174 mmol) about 45 minutes apart. The reaction mixture
was stirred for about 3 hours at about 35 °C. The solvent was then evaporated. The crude material
was purified by LC/MS to give 23 mg (32%) pure 14. ] H NMR (DMSO) 8 1.09 (t, 3H, J - 8Hz
CH3I 3.19 (m, 2H, CH 2 \ 6.79 (br s, 1H, NiT), 7.67 (d, 1H, J = 12Hz, ArH) 9 7.80 (d, 1H, J = 8 Hz,
5 AxH), 1 1.20 (br s, 1H, NH). HPLC retention time 3.58 minutes.
Example 191
1- (6-Bromo-7-(2-aminocarboxy)isopropoxy"2"benzothiazolyl)-3-ethyl"Urea (15 ): A mixture of 1-
(6-bromo-7-hydroxy-2-benzothiazoIyl)-3-ethyl-urea 6 (0.050 g, 0.16 mmol), cesium carbonate
(0.155 g, 0.48 mmol, 3.0 eq) and 60% sodium hydride (0.019 g, 0.48 mmol, 3.0 eq) in anhydrous
10 dioxane (1.0 mL) was stirred at about 20°C for about 0.5 hour, followed by the addition of 2-bromo-
2- methylpropionyl amide (0.079 g, 0.48 mmol, 3.0 eq). It was heated at about 1 10°C for about 18
hours. DMPU (2 mL) was added and it was heated at about 85°C for about another 18 hours. An
additional portion of 60% sodium hydride (0.013 g, 0.32 mmol, 2.0 eq. in minear oil) was added.
After about 3.5 days, the reaction was quenched with H2O (1 mL). The mixture was concentrated in
15 vacuum, and EtOAc (25 mL) was added. The precipitation was filtered off and was rinsed with
diethyl ether (2x5 mL) and methanol (2x5 mL). The organic solution was concentrated and
residue was dissolved in DMF (2 mL). LC/MS purification gave 0.021 g (33%) of the desired
compound 15. NMR (DMSO) 5 10.81 (br s, 1H, N#), 7.73 (br s, 1H, N#2)> 7.57 (d, 1H, J = 8.6
Hz, ArH), 7.44 (br s, 1H, Ni^X 7.34 (d, 1H, J = 8.5 Hz, ArH) 9 6.7 i (br, s, 1H; N//), 3.18 (m, 2H,
20 Ctf», 1.46 (s, 6H, CH3X 1.08 (t, 3H, J = 7.2 Hz, CH3); LC/MS 401 (MH + ); RP-HPLC RT 2.64
minutes.
6 16
Example 192
l-(6-Bromo-7-methoxy-2-benzothiazoIyl)-l"niethyI-3-ethy]"Urea (16): Compound 16 was
25 isolated in a small amount, as a side product in the formation of compound 10. 0.0030 g (2%) of the
desired compound 16 was isolated. ^H NMR (DMSO) 5 7.73 (t, 1H, J = 5.4 Hz, N#), 7.56 (d, 1H, J
= 8.5 Hz, ArH) 9 7.40 (d, 1H, J = 8.5 Hz, ArH) 9 3.94 (s, 3H, CH 3 \ 3.59 (s, 3H, CH 3 \ 3.24 (m, 2H,
CH2X 1 .09 (t, 3H, J = 7. 1 Hz, CHs)\ LC/MS 344 (MH + ); RP-HPLC RT 3.58 minutes.
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10
15
20
OH
O
8
17
Example 193
l-(7-Hydroxy-6-nitro-2-benzothiazolyi)-3-ethyl-urea (17): To a solution of 2-methylpyridone
(0.020 mL, 0.20mmol, 1.2 eq) in anhydrous acetonitrile (0.5 mL), nitronium tetrafluoroborate (0.045
g, 0.32mmol, 1 .9 eq) was added. The suspension was stirred at about 20°C for about 5 minutes to get
an orange solution. The solution was then transferred to a suspension of l-(7-hydroxy-2-
benzothiazolyl)-3-ethyl-urea 8 (0.040 g, 0.17 mmol) in acetonitrile (0.5 mL). The reaction mixture
was stirred at about 20°C for about 15 minutes, then taken up in diethyl ether (8 mL), and
neutralized by washing with saturated sodium bicarbonate solution (1.5 mL). The organic extract
was concentrated and dissolved in methanol (2 mL). LC/MS purification was followed by flash
chromatography purification on silica (methylene chloride/methanol = 40/1) to afford 0.005g (10%)
of the desired compound 17. lH NMR (DMSO) 8 1 1.20 (br s, 1H, N/7), 8.02 (d, 1H, J = 9.0 Hz,
ArH), 7.23 (d, 1H, J = 7.5 Hz, ArH), 6.78 (br s, 1H, MH), 3.20 (m, 2H 5 CH 2 \ 1.10 (t, 3 H, J = 7.2 Hz,
GHj). LC/MS 283 (MH+); RP-HPLC RT 2.74 minutes.
OR OR
6, R 1 = H
11, R 1 = CH(CH 3 ) 2
18, R 1 = H
19, R1 = CH(CH 3 ) 2
Example 194
l-(4,6-Dibromo-7-hydroxy-2-benzothiazolyl)-3-ethyl-urea, (18) : To a suspension of l-(6-bromo-
7-hydroxy-2-benzothiazolyl)-3-ethyl-urea (50 mg, 0.158 mmol) in AcOH (2 mL) at about 20°C was
added Br2 (9^L, 0.174 mmol). The reaction mixture was stirred at about 20 °C for about 15 minutes.
Toluene (10 mL) was added and the solvents evaporated. The crude material was purified by
preparative LC/MS to give 19 mg (30%) pure 18. lH NMR (DMSO) 5 1.09 (t, 3H, J = 6Hz CH 3 \
3.19 (m, 2H, CH 2 \ 6.55 (br s, 1H,N#), 7.68 (s, 1H, AxH) 9 10.51 (br s, lH,N^orOi7), 11.20(brs,
1H, Ni7or OH). HPLC retention timer 2.90 minutes.
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Example 195
l-(4,6-Dibromo-7-isopropoxy-2-benzothiazoiyI)-3-ethyl-urea, (19): Was prepared from l-(6-
bromo-7-isopropoxy-2-benzothiazolyI)-3-ethyl-urea as above. Purification by LC/MS gave 25 mg
(41 %) pure 19. *H NMR (DMSO) 5 1.08 (t, 3H, J = 8Hz, CH 2 CH 3 \ 1.32 (d, 6H, J = 4 Hz,
C\\(CH 3 ) 2 \ 3.19 (m, 2H, CH 2 \ 4.68 (m, 1H, C^(CH 3 ) 2 ) 5 6.59 (br s, 1H, Ni/) 9 7.83 (s, 1H, ArH),
1 1.45 (br s, 1H, N#). HPLC retention time 4.01 minutes.
4
and
21
10
15
20
25
Example 196-197
l.(4-ChIoro-7-hydroxy-2-benzothiazolyI)-3-ethyl-urea, (20) and l-(6-Chloro-7-hydroxy-2-
benzothiazolyI)-3-ethyl-urea, (21): To a solution of l-(7-oxo-4,5,6,7-tetrahydro-2-benzothiazolyl>
3-ethyl-urea (50 mg, 0.209 mmol) in DMF (1 mL) was added a freshly prepared solution of CI2 in
DMF (3mL) saturated at about 20 °C. The reaction mixture was stirred at about 80 °C for about 24
hours. The solvent was evaporated and the crude material was purified by LC/MS. 6 mg (1 1 %) of a
mixture of pure 20 and 21 in a ratio of 1 to 1 was obtained. *H "NMR (DMSO) 5 1.08 (t, 3H, J =
8Hz CH2CH3), 3.16 (m, 2H, CH 2 l 6.63 (d, 0.5H, J = 8 Hz, ArH) 9 6.64 (br s, 0.5H, 6.87 (br s,
0.5H, N//) 9 7.01 (d, 0.5H, J = 8 Hz, ArH), 7.22 (d, 0.5H, J = 8 Hz, ArH) 9 7.25 (d, 0.5H, J - 8 Hz,
ArH). HPLC retention time 2.54 minutes.
Example 198
l-(4,6-Dichloro-7-hydroxy-2-benzothiazoiyl)-3-ethyi-urea, (22): To a solution of l-(7-oxo-
4,5,6,7-tetrahydro-2-benzothia2olyl)-3-ethyl-urea (50 mg, 0.209 mmol) in AcOH (2 mL) was
bubbled CI2 gas for about 1 minute at about 20 °C. A white precipitate was formed that was filtered
off. 5 mg (8 %) pure 22 was obtained. ^H NMR (DMSO) 5 1.09 (t, 3H, J = 6Hz CH 3 \ 3.19 (m, 2H,
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CH2X 6.59 (br s, 1H, N//)» 1A1 ( s > 1H > Ar ^X 10 - 58 ( br s > 1H > N#or O//), 11.26 (br s, 1H, NJ7or
0#). HPLC retention time 2.73 minutes.
General Procedure for making l-(7^//cj;w);/-2-benzothiazolyl)-3-ethylurea compounds.
Step A: l-(7-TrifluoromethylsulfonyI-2-benzothiazolyI)-3-ethylurea, To a solution of 1-
5 (7-hydroxy-2-benzothiazolyl)-3-ethylurea (1.50 g, 6.32 mmol) in 15 mL pyridine at about
0°C, trifluoromethansulfonic anhydride (2.13 mL, 12.64 mmol) was added dropwise. It was
stirred at about 0°C for about 2 hours. The reaction was quenched with 1 5 mL MeOH and
the solvent was evaporated. The crude mixture was purified by flash chromatography on
Si0 2 with methylene chloride and methanol (90 / 1) to give 1.45 g (62%) of desired
10 compound. LC/MS 369.9 (M+l); LC retention time 3.34 min.
Step B: A mixture of l-(7-trifluoromethylsulfonyl-2-benzothiazolyl)-3-ethylurea,
Pd(PPh 3 ) 2 Cl 2 (0.08 eq), and triethylamine (4.3 eq) in anhydrous DMF is bubbled with
nitrogen gas for about 5 minutes, followed by the addition of an alkyne of choice (5.0 eq).
The mixture is heated at about 100°C with stirring in a sealed tube for about 18 hours. The
15 mixture is cooled down, taken up with MeOH, and evaporated to dryness. Purification by
flash chromatography on Si0 2 with ethyl acetate and heptane to give pure desired products.
Example 199
l-(7-TrimethylsiIyIacety!enyl-2-benzothiazolyl)-3-ethylurea
A mixture of l-(7-trifluoromethylsulfonyl-2-benzothiazolyl)-3-ethylurea (0.080 g, 80%
20 pure, 0.17 mmol), Pd(PPh 3 ) 2 Cl 2 (0.010 g, 0.014 mmol, 0.08 eq), and triethylamine (0.102
mL, 0.73 mmol, 4.3 eq) in 1 mL of anhydrous DMF was bubbled with nitrogen gas for 5
minutes, followed by the addition of trimethylsilyl acetylene (0.12 mL, 0.85 mmol, 5.0 eq).
The mixture was heated at about 1 00°C with stirring in a sealed tube for about 1 8 hours. The
mixture was cooled down, taken up in 10 mL MeOH, and evaporated to dryness.
25 Purification by flash chromatography on Si0 2 with ethyl acetate and heptane (2/1) gave
pure desired compound 0.050 g (93%). LC/MS 318 (M+l); LC retention time 9.25 min.
l-(7-Acetylenyl-2-benzothiazolyl)-3-ethy!urea
A mixture of l-(7-trimethylsilylacetylenyl-2-benzothiazolyl)-3-ethylurea 2 (0.050 g, 0.16
mmol) and 1M aqueous KOH solution (0.16 mL, 0.16 mmol, 1.0 eq) in 1.5 mL of 2 / 1
30 mixture of DMF / MeOH was stirred at room temperature for about 2 hours. The mixture
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was taken up in 1 0 mL MeOH and the precipitation was filtered off. The mother liquid was
concentrated and purified by prep HPLC to give the desired compound 0.006g (15%).
LC/MS 246 (M+l); LC retention time 2.77 min.
Example 200
5 i-(7-(N,N-DimethylmethylacetylenyI)-2-benzothiazolyl)-3-ethylurea
According to the general procedure for making alkynyl compounds, a mixture of l-(7-
trifluoromethylsulfonyl-2-benzothiazolyl)-3-ethylurea, triethyl amine, Pd(PPh 3 ) 2 Cl 2 , and
N,N-dimethylmethylacetylene in DMF was reacted to give 0.0025 g (8%) of the desired
compound. LC/MS 303 (M+l); LC retention time 2.20 min.
10 Example 201
l-(7-(2'-PyridinylacetylenyI)-2-benzothiazolyl)-3-ethylurea
• According to the general procedure for making alkynyl compounds, a mixture of 1 -(7-
trifluoromethylsulfonyl-2-benzothiazolyl)-3-ethylurea, triethyl amine, Pd(PPh 3 ) 2 Cl 2 , and 2-
pyridinylacetylene in DMF was reacted to give 0.020 g (57%) of the desired compound.
15 LC/MS 322.9 (M+l); LC retention time 3.18 min.
Example 202
l-(7-Isopropoxy-2-benzothiazolyl)-3-ethylurea
A solution of l-(6-bromo-7-isopropoxy-2-benzothiazolyl)-3-ethylurea (0.036 g, 0.10 mmol)
in 0.5 mL DME was cooled down to about -78°C, and was treated with a solution of 1.6 M
20 n-BuLi in hexanes (0.16 mL, 0.26 mmol, 2.6 eq). It was stirred at the temperature for about
20 min, then a solution of N-chlorosucciriimide (NCS) (0.015 g, 0.11 mmol, 1.1 eq) in 0.5
mL DME was added. It was warmed up to about 0°C for about 0.5 hour. It was taken up in
MeOH and purified by HPLC to give 0.007 g (25%) of the title compound. LC/MS 279.9
(M+l); LC retention time 3.10 min.
25 Example 203
l-(7-Phenyl-2-benzothiazolyl)-3-ethylurea
A mixture of l-(7-trifluoromethylsulfonyl-2-benzothiazolyl)-3-ethylurea (0.050 g, 80%
pure, 0.11 mmol), lithium chloride (0.039 g, 0.92 mmol, 8.4 eq), triphenylphosphine (0.017
g, 0.066mmol, 0.6 eq), Pd(PPh 3 ) 2 Cl 2 (0.010 g, .0.013 mmol, 0.12 eq), tributylphenyltin
30 (0.036 mL, 0.33 mmol, 3.0 eq), and a crystal of 2,6-di-tert-butyl-4-methylphenol in 1 mL
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anhydrous DMF was purged with nitrogen gas, and was heated at about 120°C in a sealed
tube for about 36 hours. More catalyst Pd(PPh 3 ),Cl 2 (0.010 g, 0.013 mmoL 0.12 eq) and tin
reagent (0.024 mL, 0,22 mmol, 2.0 eq) were added to the mixture after the first 24 hours.
The mixture was taken up in MeOH, filtered, and concentrated. Purification by HPLC gave
5 0.004 g (12%) of the desired compound. LC/MS 298.0 (M+l); LC retention time 3.37 min.
Example 204
l-(7-VinyI-2-benzothiazolyI)-3-ethylurea
Similar to the synthesis of Example 203, a mixture of l-(7-trifluoromethylsulfonyl-2-
benzothiazolyl)-3-ethylurea (0.050 g, 80% pure, 0.1 1 mmol), lithium chloride (0.039 g, 0.92
10 mmol, 8.4 eq), triphenylphosphine (0.017 g, 0.066mmol, 0.6 eq), Pd(PPh 3 ) 2 Cl 2 (0.009 g,
0.013 mmol, 0.12 eq), tetravinyltin (0.040 mL, 0.22 mmol, 2.0 eq), and a crystal of 2,6-di-
tert-butyl-4-methylphenol was purged with nitrogen gas, and was heated at about 1 00°C for
about 1 .5 hours. The mixture was taken up in MeOH, filtered, concentrated, and purified by
HPLC and preparative TLC in ethyl acetate / heptane mixture to obtain 0.004 g (14%) of the
15 desired compound. LC/MS 248 (M+l); LC retention time 2.96 min.
Example 205
1- (6-Bromo-7-trifluoromethylsulfonyl-2-benzothiazolyl)-3-ethyIurea
To a solution of l-(6-bromo-7-hydroxy-2-benzothiazolyl)-3-ethylurea (3.35 g, 10.6 mmol)
in 40 mL pyridine at about 0°C, trifluoromethansulfonic anhydride (2.67 mL, 15.9 mmol)
20 was added dropwise. It was stirred at about 0°C for about 4 hours. The reaction was taken up
in 100 mL AcOEt, washed with 70 mL of 2M HC1 and 70 mL of brine. The solution was
dried (MgS0 4 ) and concentrated. The crude mixture was purified by flash chromatography
on Si0 2 with AcOEt and heptane (1 / 3) to give 2.39 g (50%) of desired compound. LC/MS
445.9 (M-l); LC retention time 3.84 min.
25 l-(6j7-Di-vinyI-2-benzothiazolyl)-3-ethy!urea
Similar to the synthesis of Example 203, a mixture of l-(6-bromo-7-trifluoromethylsulfonyl-
2- benzothiazolyl)-3-ethylurea (0.100 g, 0.22 mmol), lithium chloride (0.078 g, 1.84 mmol,
8.4 eq), triphenylphosphine (0.034 g, 0.13 mmol, 0.6 eq), Pd(PPh 3 ) 2 Cl 2 (0.018 g, 0.026
mmol, 0.12 eq), tetravinyltin (0.080 mL, 0.44 mmol, 2.0 eq), and a crystal of 2,6-di-tert-
30 butyl-4-methylphenol was purged with nitrogen gas, and was heated at about 100°C for
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about 18 hours. Additional tin reagent (0.080 mL, 0.44 mmol, 2.0 eq) was added to the
mixture after the first 2 hours. The mixture was taken up in MeOH, filtered, and
concentrated. Purification by HPLC and flash chromatography on Si0 2 with ethyl acetate /
methylene chloride (1 / 4) gave the desired compound 0.014 g (23%). LC/MS 274.3 (M+l);
5 LC retention time 2.32 min.
General Procedure for making 2-amino-6~s«6s///Mte</ benzothiazole compounds.
A solution of 4-substituted aniline and KSCN (2.0 eq) in acetic acid is cooled at about 5°C,
and is treated dropwise with a solution of bromine in acetic acid (1.0 eq). The mixture is
stirred at room temperature for about 1 to 3 hours. The precipitate is filtered off and washed
10 with Et 2 0. The obtained solid is neutralized with saturated sodium carbonate solution, a new
precipitate is formed. It is filtered off, washed with water, and dried under vacuum to give
the desired compound.
Example 206
2-Amino-6-benzyl-benzothiazoIe
15 A solution of 4-benzylaniline (0.916 g, 5.00 mmol) and KSCN (0.97 g, 10.0 mmol, 2.0 eq)
in 10 mL acetic acid was cooled at about 5°C, and was treated dropwise with a solution of
bromine (0.258 mL, 5.00 mmol, 1.0 eq) in 2 mL acetic acid. The mixture was stirred at room
temperature for about 1 hour. The precipitate was filtered off and washed with Et 2 0. The
obtained solid was neutralized with saturated sodium carbonate solution, and a new
20 precipitate was formed. It was filtered off, washed with water and MeOH, and dried under
vacuum to give the desired compound 1.06 g (88%). LC/MS 241.2 (M+l); LC retention
time 2.46 min.
1 -(6-B enzy 1-2 -benzo th iazoly I)-3-ethy lu r ea
A suspension of 2-amino-6-benzyl-benzothiazole (0.040 g, 0.17 mmol), triethylamine (0.104
25 mL, 0.77 mmol, 4.5 eq), and ethyl isocyanate (0.049mL, 0.64 mmol, 3.8 eq) in 1 mL
toluene was heated at about 95°C for about 16 hours. The precipitate was filtered off,
washed with Et 2 0 and MeOH, and dried under vacuum to give the desired compound 0.029
g (56%). LC/MS 312.3 (M+l); LC retention time 2.62 min.
Example 207
30 2-Amino-6-(4'-fluorophenoxy)-benzothiazole
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A solution of 4-(4 ? -fluorophenoxy)aniline (0.305g, 1.50 mmol) and KSCN (0.29 g, 3.00
mmol, 2.0 eq) in 3 mL acetic acid was cooled at about 5°C, and was treated dropwise with a
solution of bromine (0.077 mL, 1.50 mmol, 1.0 eq) in 2 mL acetic acid. The mixture was
stirred at room temperature for about 2 hours. The precipitate was filtered off and washed
5 with Et>0. The mother liquid from the filtration was concentrated, and the residue was
combined with the precipitate. The obtained solid was neutralized with saturated sodium
carbonate solution, and a new precipitate was formed. It was filtered off, washed with water,
and dried under vacuum to give the desired compound 0.403 g (90%). LC/MS 261.2 (M+l);
LC retention time 2.44 min.
10 Example 208
2-Amino-6-(4'-pyridinylmethyl)-benzothiazole
A solution of 4-(4'-pyridinylmethyl)aniline (0.368 g, 2.00 mmol) and KSCN (0.388 g, 4.00
mmol, 2.0 eq) in 5 mL acetic acid was cooled at about 5°C, and was treated dropwise with a
solution of bromine (0.103 mL, 2.00 mmol, 1.0 eq) in 2 mL acetic acid. The mixture was
15 stirred at room temperature for 3 hours. The precipitate was filtered off and washed with
Et 2 0. The obtained solid was neutralized with saturated sodium carbonate solution, and a
new precipitate was formed. It was filtered off, washed with water, and dried under vacuum
to give the desired compound 0.41 g (85%). LC/MS 242.2 (M+l); LC retention time 1.61
min.
20 General Procedure for making l-(6-5w65//Yw^-2-benzothiazolyl)-3-ethylurea
compounds. A suspension of 2-a.mmo-6-substituted benzothiazole, triethylamine (3.0 eq)
and ethyl isocyanate (2.5 eq) in toluene is heated at about 95°C for about 3 to 20 hours. The
precipitate is filtered off, washed with Et 2 Q and MeOH, and dried under vacuum to give the
desired compound.
25 Example 209
l-(6-(4'-Fluorophenoxy)-2-benzothiazoIyl)-3-ethylurea
A mixture of 2-amino-6-(4 9 -fluorophenoxy)-benzothiazole ? triethylamine, and ethyl
isocyanate in toluene was reacted according to the general procedure described hereinabove
to give 0.041 g (66%) desired compound. LC/MS 332.2 (M+l); LC retention time 2.66 min.
30 Example 210
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l-(6-(4'-Pyridinylmethyl)-2-benzothiazolyl)-3-ethylurea
A mixture of 2-amino-6-(4'-pyridinylmethyl)-benzothiazole ? triethylamine, and ethyl
isocyanate in toluene was reacted according to the general procedure described hereinabove
to give 0.100 g (62%) of the desired compound. LC/MS 313.3 (M+l); LC retention time
1 .96 min.
Br P , HOAc F
KSCN,
Et,N, EtNCO
Toluene,
O
A
a> — N N
f H H
Example 211
l-(6-Fluoro-7-chloro-2-benzothiazolyl)-3-ethylurea
and l-(5-Chloro-6-fluoro-2-
benzothiazolyl)-3-ethylurea
10 A solution of 3-chloro-4-fiuoro-aniline (0.300 g, 2.06 mmol) and KSCN (0.412 g, 4.25
mmol, 2.06 eq) in 5 mL acetic acid was cooled to about 5°C, and was treated dropwise with
a solution of bromine (0.159 mL, 3.09 mmol, 1.5 eq) in 5 mL acetic acid. The mixture was
stirred at room temperature for about 3 hours. The mixture was concentrated and purified on
SiO, with AcOEt and methylene chloride (1 / 8) to give 0.041 g (10%) a mixture of 6-fluoro-
15 7-chloro-benzothiazole and 5-chloro-6-fluoro-benzothiazole. The mixture was mixed with
triethylamine (0.055 mL, 0.40 mmol, 2.0 eq) and ethyl isocyanate (0.031 mL, 0.40 mmol,
2.0 eq) in 1 mL toluene. It was heated at about 110°C for about one day. The residue was
evaporated to dryness, and taken up in DMF. A precipitate was filtered off from the DMF
solution, washed with Et,0, and dried under vacuum to give l-(5-chloro-6-fluoro-2-
20 benzothiazolyl)-3-ethylurea 0.003 g. LC/MS 274.0 (M+l); LC retention time 3.10 min. The
DMF mother liquid was purified by HPLC and preparative TLC to give 1 -(6-fluoro-7-
chloro-2-benzothiazolyl)-3-ethylurea 0.002 g. LC/MS 274.0 (M+l); LC retention time 3.08
min.
Example 212
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l-(6-Bromo-2-benzothiazolyl)-3-ethylurea
A suspension of 2-amino-6-bromo-benzothiazole (6.12 g, 26.2 mmol), triethylamine (730
mL, 52.4 mmol, 2.0 eq), and ethyl isocyanate (4.15mL, 52.4 mmol, 2.0 eq) in 50 mL toluene
was heated at about 90°C for about 15 hours. The precipitate was filtered off washed with
5 Et 2 0, and dried under vacuum to give the desired compound 7.39 g (94%). LC/MS 300.0
(M-l); LC retention time 3.01 min.
General Procedure for making l-(6-a//cy«j;/-2-benzothiazoIyI)-3-ethylurea compounds*
A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea ? Pd(PPh 3 ) 2 Cl 2 (0.05 eq), and
triethylamine (4.3 eq) in anhydrous DMF is bubbled with nitrogen gas for about 5 minutes,
10 followed by the addition of an alkyne of choice (5.0 eq). The mixture is heated at about
80°C with stirring in a sealed tube for about 15 hours. The mixture is cooled down, taken up
with MeOH, and evaporated to dryness. Purification by HPLC or by flash chromatography
on SiO, with ethyl acetate and heptane to give pure desired product.
Example 213
15 l-(6-TrimethylsiIylacety!enyI-2-benzothiazolyl)-3-ethylurea
A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea (0.100 g, 0.33 mmol), Pd(PPh 3 ) 2 Cl 2
(0.012 g, 0.017 mmol, 0.05 eq), and triethylamine (0.20 mL, 1.42 mmol, 4.3 eq) in 1 mL of
anhydrous DMF was bubbled with nitrogen gas for about 5 minutes, followed by the
addition of trimethylsilyl acetylene (0.23 mL, 1.65 mmol, 5.0 eq). The mixture was heated
20 at about 80°C with stirring in a sealed tube for about 15 hours. The mixture was cooled
down, taken up in 10 mL MeOH, and evaporated to dryness. Purification by HPLC gave
pure desired compound 0.093 g (89%). LC/MS 318 (M+l); LC retention time 3.77 min.
Example 214
l-(6-PhenylacetylenyI-2-benzothiazolyI)-3-ethylurea
25 A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea, Pd(PPh 3 ) 2 Cl 2 , triethylamine, and
phenylacetylene in 1 mL of anhydrous DMF was reacted according to the procedure of
Example 213 to give 0.008 g (8%) of the desired compound. LC/MS 322 (M+l); LC
retention time 3.65 min.
Example 215
30 l-(6-(N,N-Dimethylaminomenthyl)acetylenyl-2-benzothiazolyl)»3-ethylurea
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A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea, Pd(PPh 3 ) 2 Cl 2 , triethylamine, and
N,N-dimethylaminomethyl acetylene in 1 mL of anhydrous DMF was reacted according to
the procedure of Example 213 to give 0.145 g (28%) of the desired compound. LC/MS 303
(M+l); LC retention time 1.40 min.
5 Example 216
l-(6-(4 5 -Fluorophenyl)acetylenyl-2-benzothiazolyI)-3-ethylurea
A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea, Pd(PPh 3 ) 2 Cl 2 , triethylamine, and 4-
fluorophenylacetylene in 1 mL of anhydrous DMF was reacted according to the procedure of
Example 213 to give 0.215 g (94%) of the desired compound. LC/MS 340.3 (M+l); LC
10 retention time 2.92 min.
Example 217
l-(6-(4'-Tolyl)acetylenyl-2-benzothiazo!yl)-3-ethyIurea
A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea, Pd(PPh 3 ) 2 Cl 2 , triethylamine, and 4-
tolylacetylene in 1 mL of anhydrous DMF was reacted according to the procedure of
15 Example 213 to give 0.185 g (99%) of the desired compound. LC/MS 336.3 (M+l); LC
retention time 3.07 min.
Example 218
l-(6-Acetylenyl-2-benzothiazolyl)-3-ethylurea
A mixture of l-(6-trimethylsilylacetylenyl-2-benzothiazolyl)-3-ethylurea (0.0710 g, 0.22
20 mmol) and 1M aqueous KOH solution (1.22 mL, 1.22 mmol, 5.5 eq) in 1.5 mL MeOH was
stirred at room temperature for about 2 hours. The mixture acidified with 1 M HO, then
taken up in 20 mL AcOEt, and the aqueous phase was extracted with 10 mL AcOEt. The
combined organic portions were dried (MgS0 4 ), concentrated, and purified by HPLC to give
the desired compound 0.003g (5%). LC/MS 246 (M+l); LC retention time 2.84 min.
25 Example 219
l-(6-(2-PhenylethyI)-2-benzothiazolyl)-3-ethy!urea
A suspension of l-(6-phenylacetylenyl-2-benzothiazolyl)-3-ethylurea (0.048 g, 0.15 mmol)
and 10% palladium on carbon (0.016 g, 10% weight purity, 0.015 mmol, 0.10 eq) in 2 mL
ethanol was purged with nitrogen gas, followed by bubbling of hydrogen gas. It was stirred
30 under hydrogen atmosphere for about 16 hours. The mixture was taken up in 8 mL MeOH,
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filtered,- concentrated, and dried under vacuum to give 0.037 g (76%) of the desired
compound. LC/MS 326.3 (M+l); LC retention time 2.84 min.
Example 220
l-(6-(2-(4'-Fluoro-phenyl)ethyl)-2-benzothiazolyl)-3-ethylurea
A suspension of l-(6-(4'-fluorophenyl)acetylenyl-2-benzothiazolyl)-3-ethylurea (0.144 g,
0.42 mmol) and Lindlar catalyst (0.090 g, 5% weight purity, 0.042 mmol, 0.10 eq) in 8 mL
ethanol was purged with nitrogen gas, followed by bubbling of hydrogen gas. It was stirred
under hydrogen atmosphere for about 16 hours. The resulting mixture consisted of
compound having fully reduced triple bond and compound having partially reduced triple
bond. A new catalyst, palladium on carbon (0.045 g, 0.042 mmol, 0.10 eq), was added. The
reaction mixture was stirred under hydrogen gas for about another 1 hour. The mixture was
taken up in 8 mL MeOH, filtered, concentrated, and recrystallized from MeOH to give 0.046
g (38%) of the desired compound. LC/MS 344.3 (M+l); LC retention time 2.81 min.
General procedure for the synthesis of l-(6-(2(Z)-Substituted-vinyl)-2-benzothiazolyl)-3-
ethylurea compounds. A suspension of \-(6-substitued-acetylenyl-2-bQnzothiazo\y\)-3-
ethylurea compound and Lindlar catalyst (5% weight purity, 0.15 eq) in ethanol is purged
with nitrogen gas, followed by bubbling of hydrogen gas. It is stirred under hydrogen
atmosphere for about 36 hours. The mixture is taken up in MeOH, filtered, concentrated, and
purified by preparative TLC to give the desired compound.
Example 22 1
l-(6-(2(Z)-PhenyIvinyl)-2-benzothiazolyl)-3-ethylurea
A suspension of l-(6-phenylacetylenyl-2-benzothiazolyl)-3-ethylurea (0.032 g, 83% purity
by weight, 0.083 mmol) and Lindlar catalyst (0.030 g, 5% weight purity, 0.012 mmol, 0.15
eq) in 2 mL ethanol was purged with nitrogen gas, followed by bubbling of hydrogen gas. It
was stirred under hydrogen atmosphere for about 36 hours. The mixture was taken up in 10
mL MeOH, filtered, concentrated, and purified by preparative TLC to give 0.01 1 g (41%) of
the desired compound. LC/MS 324 (M+l); LC retention time 2.84 min.
Example 222
l-(6-(2(Z)-(N,N-Dimethylaminomethyl)vinyl)-2-benzothiazolyl)-3-ethylurea
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A mixture of 1 -(6-(>I ? N-dimethy lami
and Lindlar catalyst under hydrogen was reacted according to the procedure of Example 221
to give the desired compound 0.013 g (21%). LC/MS 305.3 (M+l); LC retention time 1.51
min.
5 Example 223
l-(6-(2(Z)-(4 , -FIuorophenyl)vinyl)-2»benzothiazolyl)-3-ethylurea
A mixture of l-(6-(4'-fluorophenyl)acetylenyl-2-benzothiazolyl)-3-ethylurea and Lindlar
catalyst under hydrogen was reacted according to the procedure of Example 221 to give the
desired compound 0.046g (38%). LC/MS 344.3 (M+l); LC retention time 2.81 min.
10 Example 224
l-(6-(2(Z)-(4'-Tolyl)vinyl)-2-benzothiazolyI)-3-ethylurea
A mixture of l-(6-(4'-tolyl)acetylenyl-2-benzothiazolyl)-3-ethylurea and Lindlar catalyst
under hydrogen was reacted according to the procedure of of Example 221 to give the
desired compound 0.053g (90%). LC/MS 338.3 (M+l); LC retention time 3.02 min.
15 General procedure for the synthesis of l-(6-(2(E)-5 , «to//«/^-v//2j;/)-2-benzothiazolyl)-3-
ethylurea compounds. A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea,
Pd(PPh 3 ) 2 Cl 2 (0.10 eq), l,3-bis(diphenylphosphino)propane (dppp) (0.11 eq), triethylamine
(1.2 eq), mono-substituted ethene (2.0 eq), and two crystals of BHT in anhydrous DMF is
bubbled with nitrogen gas. The mixture is heated at about 105°C with stirring in a sealed
20 tube for about 1 5 hours. The mixture is cooled down, taken up in MeOH, and evaporated to
dryness. Purification by flash chromatography on Si0 2 with MeOH and methylene chloride
yields the desired compound.
Example 225
l-(6-(2(E)-(4'-Fluorophenyl)vinyI)-2-benzothiazoiyl)-3-ethylurea
25 A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea (0.150 g 5 0.50 mmol), Pd(PPh 3 ) 2 Cl 2
(0.035g, 0.05 mmol, 0.10 eq), dppp (0.023 g, 0.055 mmol, 0.11 eq), triethylamine (0.083
mL, 0.60 mmol, 1.2 eq), 4-fluorostyrene (0.120 mL, 1.00 mmol, 2.0 eq), and two crystals of
BHT in 2 mL of anhydrous DMF was bubbled with nitrogen gas. The mixture was heated at
about 105°C with stirring in a sealed tube for about 15 hours. The mixture was cooled down,
30 taken up in 5 mL MeOH, and evaporated to dryness. Purification by flash chromatography
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on Si0 2 with MeOH and methylene chloride (1.5 / 100) gave the desired compound 0.096 g
(56%). It was further purified by precipitation from MeOH. LC/MS 342.3 (M+l); LC
retention time 2.86 min.
Example 226
5 l-(6-(2(E)-Phenylvinyl)-2-benzothiazolyI)-3-ethy!urea
A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea, Pd(PPh 3 ) 2 Cl 2 , dppp, triethylamine,
styrene, and two crystals of BHT in anhydrous DMF was reacted according to the procedure
of Example 225 to give the desired compound 0.201 g (62%). LC/MS 324.2 (M+l); LC
retention time 2.87 min.
10 Example 227
l-(6-(2(E)-(4'-Tolyl)vinyl)-2-benzothiazoIyl)-3-ethylurea
A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea, Pd(PPh 3 ) 2 Cl 2 , dppp, triethylamine,
4-methylstyrene, and two crystals of BHT in anhydrous DMF was reacted according to the
procedure of Example 225 to give the desired compound 0.030 g (37%). LC/MS 338.0
15 (M+l); LC retention time 3.80 min.
Example 228
l-(6-(2(E)-(l , -ImidazoIyl)vinyl)-2-benzothiazolyl)-3-ethylurea
A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea ? Pd(PPh 3 ) 2 Cl 2 , dppp, triethylamine,
1 -vinylimidazole, and two crystals of BHT in anhydrous DMF was reacted according to the
20 procedure of Example 225 to give the desired compound 0.308 g (97%). LC/MS 314.1
(M+l); LC retention time 1.82 min.
Example 229
Ethyl {[6-(4-Fluorophenoxy)-2-benzothiazolyl]amino}methanethioate
A suspension of 2-amino-6-(4'-fluorophenoxy)-benzothiazole (associated with 2.0 eq of KBr
25 salt, 2.50 g, 5.02 mmol) in 10 mL pyridine was treated dropwise with methyl
chlorothioformate (0.65 mL, 7.53 mmol, 01.5 eq). It was stirred at room temperature for
about 1 hour, and was poured into 40 mL ice water. The mixture was acidified slowly with 1
M HC1 solution. The result precipitate was filtered off, washed with water and MeOH, and
dried under vacuum to give 1.18 g (65%) desired compound. LC/MS 334.9 (M+l); LC
30 retention time 3.70 min.
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General procedure for the synthesis of l-(6-(4 9 -Fluorophenoxy)-2-benzothiazolyl)-3-<?/?//V?wa/(v
substituted-alkylurza compounds. A suspension of ethyl {[6-(4-fluorophenoxy)-2-
benzothiazolyI]amino}methanethioate (Example 229) and an alkyl amine (1.2 eq) of choice in EtOH
is heated at about 80°C for about 2.5 hours to 3 days. The mixture is taken up in MeOH,
concentrated, and purified by HPLC to give the desired compound.
Example 230
l-(6-(4'-FIuorophenoxy)-2-benzothiazolyl)-3-(3-(4-niethylpiperazinyI)propyl)urea
A suspension of ethyl {[6-(4-fluorophenoxy)-2-benzothiazolyl]amino}methanethioate (0.154 g, 0.46
mmol) and 4-methyl-l-(3-aminopropyl)piperazine (0.100 mL, 0.55 mmol, 1.2 eq) in 8 mL EtOH
was heated at about 80°C for about 3 days. A precipitate was filtered off, washed with MeOH, and
dried under vacuum, giving compound N-(6-(4'-Fluorophenoxy)-2-benzothiazolyl)-ethylcarbamate
as a side product. 0.004 g (3%). LC/MS 333.4 (M+l); LC retention time 3.66 min.
The mother liquid from the filtration was concentrated and purified by HPLC, it is the
desired compound in the form of the acetic acid salt. LC/MS 444.1 (M+l); LC retention time 3.32
min.
A portion of the purified compound was dissolved in methylene chloride, and washed with 2
M NaOH solution.The organic portion was evaporated, dissolved in AcOEt, and treated with 1 mL
solution of maleic acid in AcOEt. The white precipitation generated was filtered off, washed with
AcOEt, and dried under vacuum to give 0.024 g (8%) of the desired compound in the form of maleic
salt. LC/MS 444.1 (M+l); LC retention time 3.38 min.
In another run of the same reaction, the mixture was evaporated to dryness after the reaction
was finished. The residue was recrystallized from Et20 and heptane, giving 0.088 g (56%) of the
desired product as the free base. LC/MS 444.1' (M+l); LC retention time 2.17 min.
Example 23 1
l_(6-(4'-Fluorophenoxy)-2-benzothiazolyl)-3-(2-(4-imidazolyl)ethyl)urea
A mixture of ethyl {[6-(4-fluorophenoxy)-2-benzothiazoIyl]amino}methanethioate and 4-(2-
aminoethyl)imidazole in EtOH was reacted according to the procedure for obtaining the free base of
Example 230 to give the desired compound 0.027 g (46%). LC/MS 398.2 (M+l); LC retention time
2.24 min.
Example 232
l-(6-(4'-Fluorophenoxy)-2-benzothm^^
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A mixture of ethyl {[6-(4-fluorophenoxy)-2-benzothiazolyl]amino}methanethioate and 2,2-
dimethyl-3-(N,N-dimethyl)aminopropyl amine in EtOH was reacted to give the. desired compound
0.047 g (38%). LC/MS 417.2 (M+l); LC retention time 2.46 min.
The following compounds were synthesized according to the procedure for Example 230 but
using the appropriate amine:
Structure
o
o
\
N
N
'/
o
LC/MS (M+1)
518.3
518.4
517.7
502,9
413.2
LC retention time (Min)
3.53
2.96
3.92
3.88
2.22
Example 238
l_(6-(4'-Fluorophenoxy)-2-benzothiazo!yl)-3-(4-piperidinylmethyI)urea
10 A mixture of ethyl {[6-(4-fluorophenoxy)-2-benzothiazolyl]amino}methanethioate and N-Boc-4-
aminomethyl piperidine in EtOH was reacted to give the t-Boc protected analog of the title
compound as an oil. The oil was dissolved in methylene chloride at about 0°C 5 and treated with 3
mL 30% TFA solution. It was warmed up and stirred at room temperature for about 4 hours. The
mixture was concentrated, taken up in AcOEt, neutralized with NaHC03, and washed with water
15 and brine. The organic portion was concentrated and purified by HPLC to give the desired
compound 0.008 g (7% for two steps overall). LC/MS 401 .2 (M+l); LC retention time 2.26 min.
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Example 239
1- (6-(4'-Fluorophenoxy)-2-benzothiazo!yI)-3-(2-(l-piperazinyl)ethyl)urea
A mixture of ethyl {[6-(4-fluorophenoxy)-2-benzothiazolyl]amino}methanethioate 42 and l-Boc-4-
(2-aminoethyl) piperidine, and TFA was reacted according to the procedure of 48 to give the desired
5 compound 0.040g (32% for two steps overall). LC/MS 414.1 (M+l); LC retention time 2.84 min.
Example 240
2- Amino-6-aceto-benzothiazole
According to the General Procedure for 2-am\no-6-snbstituted benzothiazole compounds, a mixture
of 4-aceto-aniline ? KSCN, and bromine in acetic acid was reacted to give 6.27 g (66%) of the desired
10 compound. LC/MS 192.9 (M+l); LC retention time 2.25 min.
Example 241
l-(6-Aceto-2-benzothiazolyl)-3-ethylurea
According to the General Procedure for l-(6-5w65//n//e^-2-benzothiazolyl)-3-ethylurea compounds,
a mixture of 2-amino-6-aceto-benzothiazole 5 triethylamine and ethyl isocyanate in toluene was
15 reacted to give the desired compound 2.08 g (83%).
Example 242
Nl-Phenyl-3-(2-{[(ethylamino)carbonyl]amino}-l,3-benzothiazol-6-yI)-3-oxopropanamide
To a suspension of l-(6-aceto-2-benzothiazolyl)-3-ethylurea (0.100 g, 0.17 mmol) in 2 mL THF at
about -78°C, a solution of 1 M LiHMDS in THF (0.51 mL, 0.51 mmol, 3.0 eq) was added. It was
20 stirred at the temperature for about 15 min. The mixture was treated with phenyl isocyanate (0.022
mL, 0.20 mmol, 1.2 eq) and stirred at about -78°C for about 10 min, then warmed up to room
temperature over 5 hours. It was quenched with MeOH, slightly acidified with HC1, and
concentrated. The residue was purified by flash chromatography on SiC>2 with MeOH and
methylene chloride (1 / 100) to give the desired compound 0.002 g (3%). LC/MS 383.0 (M+l); LC
25 retention time 4. 1 0 min.
Example 243
Nl-(3-Methylphenyl)-3-(2-{[(et^
oxopropanamide
Similar to the synthesis of Example 242, a mixture of l-(6-aceto-2-benzothiazolyl)-3-ethylurea,
30 LiHMDS, and 3-methylphenyl isocyanate in THF was reacted to give 0.068 g (41%) of the desired
compound. LC/MS 396.8 (M+l); LC retention time 3.12 min.
Example 244
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Nl-[4-(Dimethylamino)phe^
oxopropanamide
Similar to the synthesis of Example 242, a mixture of l-(6-aceto-2-benzothiazolyl>3-ethylurea,
LiHMDS, and 4-dimethylaminophenyl isocyanate in THF was reacted to give 0.005 g (3%) of the
5 desired compound. LC/MS 426.1 (M+l); LC retention time 2.00 min.
Example 245
l-(6-Ethoxycarbonyl-2-benzothiazoIy!)-3-ethylurea
According to the General Procedure for l-(6-substituted-2-benzothiazoiyl)-3-ethyIurea compounds,
and similar to the synthesis of l-(6-benzyI-2-benzothiazolyl)-3-ethylurea, a mixture of 2-amino-6-
10 ethoxycarbonyl-benzothiazole, triethylamine and ethyl isocyanate in toluene was reacted to give the
desired compound 2.40 g (82%). LC/MS 294.0 (M+l); LC retention time 4.29 min.
Example 246
l-(6-(2-CyanoacetyI)-2-benzothiazo!yl)-3-ethylurea
A solution of l-(6-ethoxycarbonyl-2-benzothiazolyl)-3-ethylurea (1.20 g, 4.09 mmol) in 5 mL of
15 anhydrous DMF was treated with 2 mL of acetonitrile, cooled down to about 0°C, followed by
treatment with 1 M LiHMDS in THF (13.1 mL, 13.1 mmol, 3.2 eq). It was stirred at the temperature
for about 0.5 hour, then warmed up to room temperature, and stirred for another 4 hours. More
LiHMDS (4 mL, 4.0 mmol, 1.0 eq) was added after the first hour. The mixture was quenched with
MeOH and water, concentrated, and purified by flash chromatography on SiC>2 with MeOH and
20 methylene chloride (1 / 50) to give the desired compound 0.640g (54%). LC/MS 288.9 (M+l); LC
retention time 2.56 min.
Example 247
l-(6-(3-Aminopropanoyl)-2-benzothiazoIyl)-3-ethyIurea.
A suspension of l-(6-(2-cyanoacetyl)-2-benzothiazolyl>3-ethylurea (0.56 g, 1.94 mmol) and
25 platinum (IV) oxide (0.176 g, 0.78 mmol, 0.40 eq) in 50 mL of 2 / 3 mixture of MeOH and
chloroform was purged and bubbled with hydrogen gas. It was stirred under hydrogen gas for about
2 days. The mixture was filtered and concentrated to give 0.689 g (quantitative yield) desired
compound. LC/MS 292.9 (M+l); LC retention time 1.80 min. HPLC purification gave
corresponding acetic acid salt. LC/MS 292.9 (M+l); LC retention time 1 .80 min.
30 In a similar reaction, compound as an acetate salt form was obtained after HPLC purification.
Example 248
Nl-[3-(2-{[(Ethylamino)carbo^
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A solution of l-(6-(3-aminopropanoyl)-2-benzothiazolyl)-3-ethylurea (0.033 g, 0.10 mmol) in 1 mL
anhydrous DMF was treated with triethylamine (0.028 mL, 0.20 mmol, 2.0 eq) at room temperature.
It was stirred for about 5 min, followed by the addition of benzoyl chloride (0.014 mL 0.12 mmol,
1.2 eq). It was stirred for about 2.5 hours, quenched with MeOH, filtered, concentrated, and purified
5 by HPLC to give the desired compound 0.01 1 g (28%). LC/MS 396.7 (M+l); LC retention time 2.74
min.
Example 249
l-(6-(3-PhenylaminocarbonyIamino-propanoyI)-2-benzothiazoIyl)-3-ethylurea
A solution of l-(6-(3-aminopropanoyl)-2-benzothiazolyI)-3-ethylurea (0.033 g, 0.10 mmol) in 1 mL
10 anhydrous DMF was treated with triethylamine (0.028 mL, 0.20 mmol, 2.0 eq) at room temperature.
It was stirred for about 5 min, followed by the addition of phenyl isocyanate (0.013 mL, 0.12 mmol,
1.2 eq). It was stirred for about 2.5 hours, quenched with MeOH, filtered, concentrated, and purified
by HPLC to give the desired compound 0.009 g (22%). LC/MS 412.2 (M+l); LC retention time 2.80
min.
15 Example 250
l-(6-(3-(3-MethyIphenyI)aminocarbonylamino-propanoyI)-2-benzothiazoIyl)-3-ethylurea
Similar to the synthesis of Example 249, a mixture of l-(6-(3-aminopropanoyI)-2-benzothiazolyl)-3-
ethylurea, triethylamine and 3-methylphenhyl isocyanate in DMF was reacted to give the desired
compound 0.021 g (25%). LC/MS 426.1 (M+l); LC retention time 2.94 min.
20 Example 251
Nl-[3-(2-{[(Ethylamino)carbonyI]amino}-l,3-benzothiazol-6-yl)-3-oxopropyI]-4-
(dimethylamino)benzamide
Similar to the synthesis of Example 248, a mixture of l-(6-(3-aminopropanoyl)-2-benzothiazolyI)-3-
ethylurea, triethylamine and 4-dimethylaminobenzoyl chloride in DMF was reacted to give the
25 desired compound 0.025g (25%) as an acetic acid salt. LC/MS 440.1 (M+l); LC retention time 2.86
min.
Example 252
Nl-[3-(2-{[(EthyIamino)carbonyl]amino}-l,3-benzothiazoI-6-yI)-3-oxopropyI]-4-
fluorobenzamide
30 Similar to the synthesis of Example 248, a mixture of l-(6-(3-aminopropanoyl)-2-benzothiazolyl)-3-
ethylurea, triethylamine and 4-fluorobenzoyl chloride in DMF was reacted to give the desired
compound 0.008 g (10%). LC/MS 415.1 (M+l); LC retention time 2.17 min.
Example 253
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Nl-[3-(2-{[(Ethylamino)carbonyI]amino}-l,3-benzothiazol-6-yl)-3-oxopropyl]-2,5-
difluorobenzamide
Similar to the synthesis of Example 249, a mixture of l-(6-(3-aminopropanoyI)-2-benzothiazolyl>3-
ethylurea, triethylamine and 2,5-difluorobenzoyl chloride in DMF was reacted to give the desired
5 compound 0.019 g (22%). LC/MS 433.0 (M+l); LC retention time 2.94 min.
Example 254
l-(6-Bromo-l,3-benzothiazol-2-yI)-3-ethyl-5-methyl-l,3 9 5-triazinan-2-one*
A mixture of l-(6-bromo-2-benzothiazolyl)-3-ethylurea ? methylamine, formaldehyde, and
N-methyl morpholine in a mixed solvent of ethanol and water was reacted to give the
10 desired compound 1 .56 g (88%).
Example 255
l-(6-(2(E)-(Ethoxycarbonyl)vinyl)-2-benzothiazolyl)-3-ethylurea
According to the general procedure described for the synthesis of 1 -(6-(2(E)-substituted-
vinyl)-2-benzothiazolyl)-3-ethylurea compounds, a mixture of l-(6-bromo-l,3-benzothiazol-
15 2-yl)-3-ethyl-5-methyl-l ? 3 ? 5-triazinan-2-one, Pd(PPh 3 ) 2 Cl 2? dppp, triethylamine, ethyl
acrylate, and two crystals of BHT in anhydrous DMF was reacted to give the desired
compound 65 0.022 g (29%). LC/MS 375.0 (M+l); LC retention time 3.63 min, A solution
of compound (0.019 g, 0.051 mmol) in 1 mL 4 M HC1 in dioxane was stirred at room
temperature for about 4 hours. The mixture was filtered off, and the solid was washed with
20 MeOH and dried under vacuum to give the desired compound 66 0.015 g (94%). LC/MS
320.2 (M+l); LC retention time 2.23 min.-
Example 256
l„(6-(3-Aminophenyl)-2-benzothiazolyl)-3-ethylurea.
A suspension of l-(6-bromo-2-benzothiazolyl)-3-ethylurea (0.300 g, 1.00 mmol), 3-
25 aminophenyl boric acid (0.237 g, 1.50 mmol, 1.5 eq) 5 and sodium bicarbonate (0.210 g, 2.50
mmol, 2.5 eq) in 8 mL of mixed solvent DMF / water (5 / 1) was purged with nitrogen gas.
To the mixture, the catalyst Pd(PPh 3 ) 4 (0.058 g, 0.05 mmol, 0.05 eq) was added. It was
purged with nitrogen gas again, and was heated at about 100°C in a sealed tube for about 48
hours. More Pd(PPh 3 ) 4 (0.025 g, 0.02 mmol, 0.02 eq) and more boric acid (0.080 g, 0.50
30 mmol, 0.50 eq) were added after the first 24 hours. The mixture was taken up in MeOH,
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concentrated, dissolved in methylene chloride, washed with sodium bicarbonate solution,
dried (MgS0 4 ), evaporated, and purified by flash chromatography on SiO, with MeOH and
methylene chloride (1 / 50) to give the desired compound 0.073 g (23%). LC/MS 313.2
(M+l); LC retention time 2.1 1 min.
5 Example 256
l-(6-(3-Phenylaminocarbonylaminophenyl)-2-benzothiazolyl)-3-ethylurea
A suspension of l-(6-(3-aminophenyl)-2-benzothiazolyl)-3-ethylurea (0.062 g, 0.20 mmol),
triethylamine (0.083 mL, 0.60 mmol, 3.0 eq), and phenyl isocyanate (0.055 mL, 0.50 mmol,
2.5 eq) in 2 mL toluene was stirred at room temperature for about 1 hour. The white
1 o precipitation was filtered off, washed with Et 2 0 and MeOH, and dried under vacuum to give
the desired compound 0.038 g (44%). LC/MS 431.8 (M+l); LC retention time 3.49 min.
Example 257
4-Pyrazolylboronic pinacolate
A mixture of 4-bromopyrazole (3.02 g, 20.3 mmol), bis(pinacolato) diborane (6.20 g, 24.4
15 mmol, 1.2 eq), KOAc (5.99 g, 60.9 mmol, 3.0 eq), and catalyst Pd(dppf)Cl 2 /CH 2 Cl 2 (0.83 g,
1.02 mmol, 0.05 eq) in 40 DMF was purged with nitrogen gas. It was heated at about 95°C
in a sealed bottle for about 1 5 hours. It was taken up in AcOEt, concentrated, taken up again
in AcOEt, filtered through a silica gel column, and concentrated. The residue was further
purified by flash chromatography on SiO, with EtOAc and heptane (1 / 1) to give the desired
20 compound 2.46 g (62%). LC/MS 1 95.1 (M+l); LC retention time 1 .59 min.
Example 258
l_(6-(4-Pyrazolyl)-2-benzothiazolyl)-3-ethylurea
Similar to the synthesis of l-(6-(3-aminophenyl)-2-benzothiazolyl)-3-ethylurea, a mixture of 4-
pyrazolylboronic pinacolate, l-(6-bromo-2-benzothiazolyl)-3-ethylurea, sodium carbonate, and
25 Pd(PPli3)4 in DMF / water mixed solvent was reacted to give the desired compound 0.543 g (30%).
LC/MS 288.2 (M+l); LC retention time 2.61 min.
Example 259
l-(6-(Pinacolatoborano)-2-benzothiazolyl)-3-ethylurea
Similar to the synthesis of 4-pyrrazolylboronic pinacolate, a mixture of l-(6-bromo-2-
30 benzothiazolyl)-3-ethylurea, bis(pinacolato) diborane, KOAc, and catalyst Pd(dppf)Cl 2 /CH2Cl2 in
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DMF was reacted to give the desired compound 3.31 g (93%). LC/MS (M+l) 348.1; LC retention
time 2.62 min.
Example 260
Methyl 4-(2-{[(ethylamino)carbonyI]amino}-l,3-benzothiazoI-6-yI)-li?-2-pyrrolecarboxylate
5 Similar to the synthesis of l-(6-(4-pyrrazolyl)-2-benzothiazolyl)-3-ethylurea, a mixture of l-(6-
(pinacolatoborano)-2-benzothiazolyl)-3-ethylurea, methyl 4-bromopyrrole-2-carboxylate, sodium
carbonate, and Pd(PPh3>4 in DMF / water mixed solvent was reacted to give the desired compound
0.001 g (1%). LC/MS 345.0 (M+l); LC retention time 3.04 min.
Example 261
10 l-(6-(4-Chlorophenyi)-2-benzothiazolyI)-3-ethylurea
Similar to the synthesis of l-(6-(4-pyrrazolyl)-2-benzothiazolyl)-3-ethylurea, a mixture of l-(6-
bromo-2-benzothiazolyl)-3-ethylurea, 4-chlorophenylboric acid, sodium bicarbonate, and Pd(PPh3)4
in DMF / water mixed solvent (5/1) was reacted to give the desired compound 0.020 g (18%).
LC/MS 330 (M-l); LC retention time 2.70 min.
15 Example 262
l-(6-(3-(3-TolyI)aminocarbonylaminophenyI)-2-benzothiazolyI)-3-ethylurea
Similar to the synthesis of l-(6-(3-phenylaminocarbonylaminophenyl)-2-benzothiazolyl)-3-
ethylurea, a mixture of l-(6-(3-aminophenyl)-2-benzothiazolyl)-3-ethylurea ? triethylamine,
and 3-tolyl isocyanate in toluene was reacted to give the desired compound 0.023 g (26%).
20 LC/MS 446.2 (M+l); LC retention time 3.76 min.
Example 263
l-(6-(l-Phenylaminocarbonylpyrrazol-4-yl)-2-benzothiazolyl)-3-ethylurea
Similar to the synthesis of l-(6-(3-phenylaminocarbonylaminophenyl)-2-benzothiazolyl)-3-
ethylurea, a mixture of l-(6-(4-pyrrazolyl)-2-benzothiazolyl)-3-ethylurea ? triethylamine, and
25 phenyl isocyanate in toluene was reacted to give the desired compound 0.025g (23%).
LC/MS 407.1 (M+l); LC retention time 3.85 min.
Example 264
l-(6-(3-(2-Fluorophenyl)aminocarbonylamino-propanoyI)-2-benzothiazoIyl)-3-ethyIurea
Similar to the synthesis of Example 249, a mixture of l-(6-(3-aminopropanoyl)-2-benzothiazolyl)-3-
30 ethylurea, triethylamine and 2-fluorophenyl isocyanate in DMF was reacted to give the desired
compound 0.022g (26%). LC/MS 430.01 (M+l); LC retention time 2.89 min.
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Example 265
l-(6-(4 , -Fluorophenoxy)-2-benzothiazolyl)-3-(2-(3-methylaminopropyl))urea
Similar to the synthesis of l-(6-(4 , -fluorophenoxy>2-benzothiazolyl)-3-(4-piperidinylmethyl)urea ? a
mixture of ethyl {[6-(4-fluorophenoxy)-2-benzothiazolyl]amino}methanethioate ? 3-Boc-3-
methylpropylamine, and TFA was reacted to give the desired compound 0.044g (39% for two steps
overall). LC/MS 375.0 (M+l); LC retention time 3.17 min.
R1
General procedure for making a compound of the formula
A suspension of AH6-bromo-7-oxo-4,5,6,7-tetrahydro-l ^-benzothiazol^-yO-TV-ethylurea and a
thioamide of choice (1 eq.) in n-propanol is heated to about 105 °C for about 16 hrs. The reaction
mixture is concentrated in vacuo and the residual crude material is purified by preparative HPLC.
Example 266
Ar-Ethyl-AM7-(3-pyridyO
A suspension of ^.(6-bromo-7-oxo-4,5 ; 6,7-tetrahydro-l,3-benzothiazol-2-yl>A^-ethylurea (25 mg,
0.079 mmol) and thionicotinamide (1 1 mg, 0.079 mmol) in n-propanol (0.4 mL) was heated to about
105 °C for about 16 hrs. The reaction mixture was concentrated in vacuo and the residual crude
material was purified by preparative HPLC. 10 mg (36%) pure product was isolated.
Example 267
AMEthyl-A^7-ethyl^
As above but using the appropriate starting material instead of thionicotinamide. 3 mg (20%) product
was isolated.
R1
General procedure for making a compound of the formula
A suspension of
yl]urea and DDQ (2 eq.) in toluene is heated to about 35 °C for about 3 hrs. The reaction mixture is
> concentrated in vacuo and purified by preparative HPLC.
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Example 268
7V-Ethyl-A^~[7-(3-pyri^
A suspension of AT-ethyI-/^[7K3-pyridyl)^
2-yl]urea (10 mg, 0.028 mmol) and DDQ (13 mg, 0.056 mmol) in toluene (1 mL) was heated to
5 about 35 °C for about 3 hrs. The reaction mixture was concentrated in vacuo and purified by
preparative HPLC. 3 mg (30 %) pure product was isolated. LC/MS 356 (MH + ); RP-HPLC 14.25
min.
Example 269
A/-Ethyl-A^7-e^
10 As above but using N-ethyl-N'-(7-ethyl-4 5 5-dihydro[l,3]thiazolo[4' ? 5':3 > 4]benzo[d]-[l 5 3]thiazol-z-
yl)urea as the starting material. 16 mg (23 %) product was isolated. LC/MS 307 (MH + ); RP-HPLC
2.88 min.
R2
s'
15 General procedure for making a compound of the formula
A suspension of A^-(6-bromo-7-oxo-4 5 5 ? 6 5 7-tetrahydro-l,3-benzothiazol-2-yl)-A^-ethylurea and a
thioamide of choice (1 eq.) in THF is heated to about 65 °C for about 1.5 hrs. The reaction mixture is
pumped down and used in the next step without further purification
Example 270
20 jV-[7-(4-Bromoanilino^
ethylurea
A suspension of A?-(6-bromo-7-oxo-4 5 5 ? 6 5 7-tetrahydro-l s 3-benzothiazol-2-yl)-A^-ethylurea (50 mg,
0.16 mmol) and 4-bromo-phenyI-thiourea (36 mg, 0.16 mmol) in THF (1 .0 mL) was heated to about
65 °C for about 1 .5 hrs. The reaction mixture was pumped down and used in the next step without
25 further purification.
Example 271
AqEthyl-iV-(7-piperidm
As above but using piperidylthiourea.
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Example 272
As above but using methylthiourea.
5 General procedure for making a compound of the formula
To a suspension of 7V-[7-( J R2)-4 5 5-dihydro[l,3]thiazolo[4 , ,5 , :3,4]benzo[<] [l,3]thiazol-2-yl]-iV-
ethylurea in toluene is added DDQ (2 eq.). The reaction mixture is stirred at about 20 °C for about 2
hrs. The reaction mixture is pumped down in vacuo and purified by preparative HPLC
Example 273
10 Aq7-(4-Bromoanilino)[13]thiaz^
To a suspension of A^47-(4-bromoanilino)-4 ; 5-dihydro[l ? 3]thiazoIo[4 , ? 5':3 ? 4]benzo[a r l [l,3]thiazol-2-
yl]-yY-ethylurea (27 mg, 0.060 mmol) in toluene (2.0 mL) was added DDQ (28 mg, 0.125 mmol).
The reaction mixture was stirred at about 20 °C for about 2 hrs. The reaction mixture was pumped
down in vacuo and purified by preparative HPLC. 7 mg (26%, 2 steps) pure product was isolated.
15 LC/MS 448 and 450 (MH + ); RP-HPLC 18.09 min.
Example 274
AT-EthyI-A^7-piperidino[^
As for Example 273 but using the appropriate starting material . 18 mg (21 %, 2 steps). LC/MS 362
(MH + ); RP-HPLC 16.97 min.
20 Example 275
AT-Ethyl-AM7-(l-methylammonto^^ acetate
As for Example 273 but using the appropriate starting material. 5mg ( 7%, 2 steps) of pure product
was isolated. LC/MS 308 (MH + ); RP-HPLC 8.75 min.
Example 276
25 2-(3-Ethyl-5-methyI-2-oxo-l,3^^
To a suspension of A^-ethyl-TV^T-oxo^^^J-tetrahydro-l^-benzothiazol^-yOurea (10.0 g, 41.79
mmol) in EtOH/H20 (1/1, 300 mL) were added formaldehyde (37 wt % in H2O, 20.4 g 5 417.87
mmol), methylamine (40 wt % in H2O, 10.8 mL, 125.37 mmol) and N-methylmorpholine (1 1.8 mL,
83.6 mmol). The suspension was heated to about 60 °C for about 5 hrs, then a solution had been
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formed. The reaction mixture was pumped down in vacuo to give 12.41 g (quantitative yield) pure
product.
Example 277
2-(3-Ethyl-5-methyK2-oxo-l,3,5-triazinan-l-yI)-6-[(Z)-l-hydroxymethylide
tetrahydro-l,3-benzothiazoI-7-one
To a suspension of 2-(3-ethyl-5-methyl-2-oxo-l ; 3 5 5-triazinan-l-yl)-4 5 5 ? 6 9 7-tetrahydro-l,3-
benzothiazoI-7-one (2.0 g ? 6.79 mmol) and ethylformate (2.5 mL, 30.95 mmol) in toluene (60 mL) at
about 0 °C was added solid NaH (60% in mineral oil, 2.5g, 30.48 mmol). The reaction mixture was
stirred at about 0°C for about 7 hrs then poured into saturated NH4CI aq. The product was extracted
into CH2CI2 (3 times). The combined organic phases were filtered and the filtrate concentrated to
give 2.5 g of crude product that was used in the next step without further purification.
General procedure for making a compound of the formula
To a suspension of 2-(3~ethyl-5-methyl-2-oxo-l ? 3 3 5-triazinan-l-yI)-6-[(Z)-l-hydroxymethylidene]-
4,5,6,7-tetrahydro-l,3-benzothiazol-7-one in EtOH is added R2HNNH2* XH2O (4 eq.). The reaction
mixture is stirred at about 20 °C for about 16 hrs, additional R2HNNH2-XH2O (4 eq.) is added and
the reaction mixture stirred at about 40 °C for about another 4 hrs, still some unprotected product
present. A third portion of R2HNNH2*XH20 (8 eq.) is added and the reaction mixture stirred for
about another 7 hrs at about 45 °C. The reaction mixture is pumped down and carried on to the next
step without further purification. An analytically pure sample is achieved by preparative HPLC.
To a suspension of 2-(3-ethyl-5-methyl-2-oxo-l,3,5-triazinan-l-yl)-6-[(Z)-l-hydroxymethylidene]-
4,5,6,7-tetrahydro-l,3-benzothiazol-7-one (100 mg, 0.31 mmol) in EtOH (2.5 mL) was added
H2NNH2- XH2O (0.040 mL, 1.24 mmol). The reaction mixture was stirred at about 20 °C for about
16 hrs, additional H2NNH2*XH20 (0.040 mL, 1.24 mmol) was added and the reaction mixture
stirred at about 40 °C for about another 4 hrs, still some unprotected product present. A third portion
of H2NNH2-XH20 (0.080 mL, 2.48 mmol) was added and the reaction mixture stirred for about
another 7 hrs at about 45 °C. The reaction mixture was pumped down and carried on to the next step
without further purification. An analytical pure sample was achieved by preparative HPLC. LC/MS
264 (MH + ); RP-HPLC 9.72 min.
R2
Example 278
A^-(5,8-Dihydro-4JT-[l,3]thiazolo[4,5-g]indol-2-yl)-7V , -ethylurea
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5
Example 279
A^Ethyl-A"-(8-meth^
As above but using MeHNNFT?.* XH2O rather than H2NNH2* XH2O. This product was carried on
to the next step without extensive purification at this stage.
General procedure for making a compound of the formula
To a suspension of A^(5,8-dihydro-4/f-[l,3]thiazo in toluene is
added DDQ (1.1 eq.). The reaction mixture is stirred at about 45°C for about 4 hrs. The reaction
mixture is pumped down and the crude material is purified by preparative HPLC.
10 Example 280
iV-EthyI-A^-(8J7-[l,3]thiazolo[4 ? 5-^]indol-2-yI)urea
To a suspension of iV-(5,8-dihydro-4//-[U3]thiazolo[4,5-g]indol-2-yl)-7^-ethylurea (81 mg, 0.308
mmol) in toluene (3.5 mL) was added DDQ (78 mg, 0.34 mmol). The reaction mixture was stirred at
about 45°C for about 4 hrs. The reaction mixture was pumped down and the crude material was
15 purified by preparative HPLC. 3 mg (4 %, 2 steps) pure product was isolated. LC/MS 262 (MH + );
RP-HPLC RT 10.45 min.
Example 281
7-[(Ethylamino)carbonyl]amino-l-methyI-m-[l,3]thiazolo[4,5-g]indazol-l-ium acetate
As described for Example 280 but using the appropriate starting material. 6 mg (20 %, 2 steps) of
20 pure product was isolated. LC/MS 276 (MH + ); RP-HPLC 1 1 .70 min.
Example 282
A^7J-Di(phenyIsulfanyl)-4,5,6/7-tetrah
A suspension of ^-ethyl-7V'-(7-oxo-4 ? 5 5 6,7-tetrahydro-l 3 3-benzothiazol-2-yl)urea (1.0 g, 4.18
mmol) and thiophenol (0.59 mL, 4.74 mmol) in EtOH (20 mL) was saturated with HC1 (g) at about 0
25 °C. The reaction mixture was stirred at about 20 °C for about 2 hrs then a second portion of
thiophenol (0.59 mL, 4.74 mmol) was added and the reaction mixture stirred for about another 2 hrs.
The ethanol was removed in vacuo and H2O was added. The aqueous phase was neutralized by
addition of 2M NaOH and the product was extracted into CH2O2 (4 times). The combined organic
phases were dried over MgSC>4. Evaporation of the solvent gave L85g (quantitative yield) pure
30 product.
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Example 283
7V-EthyI-A^-[7-(phenylsulfanyl)-4,5-dihydro-l,3-benzothiazol-2-yl]urea^
To a suspension of Aq7,7^i(phenylsuIfanyI)-4^
(700 mg, 1.59 mmol) in THF (14.0 mL) was added DBU (0.36 mL, 2.38 mmol). The reaction
mixture was stirred at about 20 °C for about 30 min. The reaction was concentrated in vacuo. The
residual yellow oil was taken up in CH2CI2 , washed with ACOH/H2O 1/10 and dried over MgSC>4.
The organic solvent was removed in vacuo and the residual oil (l.Og) was used in the next step
without further purification.
Example 284
iV-EthyI-A^-[7-(phenylsulfanyl)-l,3-benzothiazol-2-yl]urea
To a mixture of 7V-ethy]-7V T -[7-(phenylsulfanyl)-4 3 5-dihydro-l ? 3-benzothiazol-2-yl]urea (crude
product from the previous reaction, 1.59 mmol) in toluene (35.0 mL) was added DDQ (500 mg, 2.17
mmol). The reaction mixture was stirred at about 20 °C for about 2 hrs. The toluene was removed in
vacuo and the crude material was taken up in CH 2 C\2- The organic phase was washed with 0.5 M
NaOH (2 times) and dried over MgSC>4. The crude oil was purified by flash chromatography on
Si02 (EtOAc/CH 2 Cl2 5/95). 445 mg (85 %, 2 steps) of pure product was isolated. LC/MS 330
(MH + ); RP-HPLC RT 17.56 min.
Example 285
A r -Ethyl-A^-[7-(phenylsulfinyI)-l,3-benzothiazol-2-yI]urea
To a suspension of A^ethyl-T^-p^phenylsulfanyO-l^-benzothiazol^-ylJurea (107mg, 0.32 mmol)
in CH 2 Cl2 (5 mL) was added MCPBA (60 mg, 0.24 mmol, 70%). The reaction mixture was stirred
at about 20 °C for about 1.5 hrs. The reaction mixture was then concentrated in vacuo. The crude
reaction mixture was purified by flash chromatography on SiC>2 (EtOAc/CH2Cl2 10/90). 32 mg
(29%) of pure product was isolated. LC/MS 346 (MH+); RP-HPLC RT 12.96 min.
Example 286
A r -Ethyl-A^-[7-(phenylsuIfonyl)-l ? 3-benzothiazol-2-yl]urea
To a suspension of A^ethyl-7V T -[7-(phenylsulfanyl)-l,3-benzothiazol-2-yl]urea (150 mg, 0.46 mmol)
in CH2CI2 (5 mL) was added MCPBA (125 mg, 0.50 mmol, 70%). The reaction mixture was stirred
at about 20 °C for about 1.5 hrs then more MCPBA ( 60 mg, 0.24 mmol, 70%) was added. The
reaction mixture was stirred for about 1 more hour then concentrated in vacuo. The crude reaction
mixture was purified by flash chromatography on Si0 2 (EtOAc/CH 2 Cl 2 10/90). 45 mg (27%) of
pure product was isolated. LC/MS 362 (MH + ); RP-HPLC RT 34.41 min.
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10
Example 287
A^-(6-Bromo-l^-benzothiazol-2-yl)"A^-(3-chloropropyl)urea
See general procedure below
General procedure for the preparation of a compound of
the formula
(See Table 1).
To a mixture of A^(6-bromo-l,3-benzothiazol-2-yl)-A^-(3-chloropropyl)urea in THF/EtOH is added
an amine of choice (10 eq.). The reaction mixture is heated to about 55 °C for about 16 hrs. The
crude reaction mixture is pumped down and purified by flash chromatography on Si02-
Example 289
iV-(6-Bromo-l,3-benzothiazol-2-yl)-7V-(3-piperazinopropyl)urea
To a mixture of A^^-bromo-US-benzothiazol^-yO-A^^S-chloropropyOurea (100 mg, 0.287 mmol)
in THF/EtOH (0.50/0.25 mL) was added piperazine (247 mg, 2.87 mmol). The reaction mixture was
heated to about 55 °C for about 16 hrs. The crude reaction mixture was pumped down and purified
by flash chromatography on Si02-
15
Table 1
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Example
R2
RP-HPLC
RT (min.)
LC/MS
(MH+)
287
C!
16.78
349/351
288
boc
14.7
510/512
289
O
N
H
12.1
398/400
290
cr
H
12.54
426/428
291
Xx
H
12.51
426/428
292
O
N NH 2
*
1 1 .96
4 OO/440
293
11.85
426/428
294
11.89
413/415
295
Q
OH
11.93
399/401
General procedure for the preparation of a compound of the formula
O
o
A.
/) — N N
H H
OH
, where Rl is Br (See Table 2).
To a solution of ethyl 4-([(6-bromo-l,3-benzothiazol-2-yl)amino]carbonylamino)-butanoate in
THF/EtOH is added 2 M NaOH (10 eq.). The reaction mixture is stirred at about 20 °C for about 3
hrs then 2 M HC1 is added until pH < 6 and a white precipitate is formed. The precipitate is filtered
off and washed with H2O, pure product is isolated.
Example 299
4-([(6-Bromo-l,3-benzothiazol-2-yl)amino]carbonyIamino)butanoic acid
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To a solution of ethyl 4-([(6-bromo«l,3-benzothiazoI-2-yl)amino]carbonylamino)-butanoate (175
mg, 0.453 mmol) in THF/EtOH (1/1, 3 mL) was added 2 M NaOH (2.26 mL, 4.53 mmol). The
reaction mixture was stirred at about 20 °C for about 3 hrs then 2 M HC1 was added until pH < 6 and
a white precipitate was formed. The precipitate was filtered off and washed with H2O. 25mg (15%)
5 pure product was isolated.
Example 306
4-([(6-Chloro-l,3-benzothiazol-2-yI)amino]carbonyIamino)butanainide
Gaseous ammonia was bubbled through a suspension of ethyl 4-([(6-chloro-l ? 3-benzothiazol-2-
yl)amino]carbonylamino)butanoate (155 mg, 0.453 mmol) in MeOH (1.5 mL) for about 5 min the
10 reaction mixture was then heated to about 85 °C for about 2 hrs. This procedure was repeated 4
times. The reaction mixture was cooled down and the white ppt was filtered off. 62 mg (44%) pure
product was isolated.
General procedure for the preparation of a compound of the formula
O O
— N^N^><^R2
H H
where Rl is Br and R2 is an amine (see Table 2).
15 Ethyl 4-([(6-bromo-l,3-benzothiazol-2-yl)amino]carbonylamino)butanoate is heated neat at about 80
°C with an amine of choice (10 eq.) for about 8 hrs. To the crude reaction mixture is added THF and
the precipitate is filtered off and washed with THF
Example 298
7V-(6-Bromo-l,3-benzothiazol-2-yl)-A^-[4-(4-methylpiperazino)-4-oxobutyl]urea
20 Ethyl 4-([(6-bromo-I,3»benzothiazol-2-yI)amino]carbonylamino)butanoate (100 mg, 0.259 mmol)
was heated neat at about 80 °C in A^-methyl-piperazine (259 mg, 2.59 mmol) for about 8 hrs. To the
crude reaction mixture was added THF (2 mL), the ppt was filtered off and washed with THF (1
mL). The product was dried in vacuo and gave 67 mg (53 %) pure product.
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Example
R1
n
Compound
R2
RP-HPLC
RT (min.) |
LC/MS
(MH+)
296
Br
2
33
o
N
I
12.14
440/442
297
Br
2
34
OH
13.02
358/360
298
Br
2
I
35
12.27
470/472
299
Br
2
36
12.76
468/470
300,
Br
2
37
It
H
12.18
454/456
301
CI
2
38
c
I
8.59
426
302
CI
2
39
8.45
424
303
CI
2
40
^ —
424
304
CI
2
41
NH2
12.32
313
305
Ci
2
42
K
NH
17.25
436
306
CI
1
43
OH
12.19
300
307
CI
1
44
o
15.57
367
308
CI
1
45
r
11.83
398
309
CI
1
46
11.73
412
310 .
Ci
1
47
12.2
410
311
CI
1
48
t
11.73
382
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312
CI
1
49
NH
16.57
422
313
p-F-PhO
2
50
O
N
I
14.03
472
Example 314
A^ie-CS-Chloro-l-thienyO-l^-benzothiazol-l-yll-TV-ethylurea
5 A 40 ml pressure tube was charged with about 100 mg A ^ -(6-bromo-l,3-benzothiazol-2-yl)-A/ , -
ethylurea, and about 2 ml ethylene glycol dimethyl ether. The slurry was stirred via magnetic stirbar
followed by evacuation then release to nitrogen atmosphere (3 times). Next about 6 mol percent
Pd(PPh3)4 was added, followed by about 1.1 eq of 5-chlorothiophene-2-boronic acid. Following
addition of about 3 equivalents sodium carbonate in about 0.5 ml water, the suspension was
3 0 evacuated and released to nitrogen atmosphere 3 times. The pressure tube was then sealed tightly
and heated to about 85-90°C for about 12-20 hours. The reaction was cooled to room temperature
then purified by preparative HPLC to yield 16% of A^-[6-(5-chloro-2-thienyl)-l,3-benzothiazol-2-yl]-
iV-ethylurea. *H NMR 1.1 (t, 3H), 3.2 (m, 2H), 6.75 (m ? 1H), 7.18 (d, 1H), 7.38 (m, 1H), 7.6 (m,
2H) 5 8.19 (d ? 1H) ? 10.75 (br s 5 1H); LC/MS 3.86 min, 338 (M+l), 336 (M-l).
15 Example 3 1 5
iV-[6-(5-Chloro-2-thienyl)-l,3-benzothiazoI-2-yl]-iV-ethylurea
This is an alternative procedure for making the compound of Example 314. A 40 ml pressure tube
was charged with about 32 mg 5-chlorothiophene-2-boronic acid , about 50 mg 7V-(6-bromo-l,3-
benzothiazol-2-yl)-A^-ethylurea and about 30 mg potassium fluoride as base. About 3 ml ethylene
20 glycol dimethyl ether was added and the suspension was vacuum purged and released to nitrogen
three times. The catalyst solution was prepared in a separate flask as follows: about 31 mg of
Pd(OAc)2 and about 110 mg of 2-dicyclohexylphosphino-2'-(N 5 N-dimethyiamino)biphenyl were
charged followed by about 6 ml ethylene glycol dimethyl ether. The flask was vacuum purged and
released to nitrogen 3 times followed by stirring to complete solution. An appropriate amount of
25 catalyst solution (about 10-50 mol percent catalyst) was then transferred to the pressure tube. The
pressure tube was then evacuated then released to nitrogen atmosphere three times, sealed tightly
and heated to about 85-90°C for about 12-20 hours. Upon cooling to room temperature the now clear
solution was purified by preparative HPLC to yield about 33% of JV-[6-(5-chloro-2-thienyl)-l,3«
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benzothiazoI-2-yI]-A^-ethyiurea. 'HNMR LI (t, 3H), 3.2 (m, 2H), 6.75 (m, 1H), 7.18 (d, 1H), 7.38
(m, 1H), 7.6 (m, 2H), 8.19 (d, 1H), 10.75 (br s, 1H); LC/MS 3.86 min, 338 (M+l), 336 (M-l).
Example 3 1 6
7V-[6-(5-Chloro-2-thienyl)-l,3-benzothiazol-2-yl]-iV-ethylurea
This is another alternative method of making the compound of Example 314. A 40 ml pressure tube
was charged with about 100 mg of ^-(e-bromo-l^-benzothiazol^-yO-A^-ethylurea, about 1.2
equivalents pinacoldiborane, about 3 equivalents potassium acetate and about 1.5 ml
dimethylformamide. The reaction mixture was evacuated and released to nitrogen 3 times. About 5
mole percent of PdC^dppf was added and the reaction flask was again evacuated and released to
nitrogen 3 times. The tube was tightly sealed and the mixture was heated to about 85-90°C overnight.
Following cooling to room temperature, the mixture was purified on silica gel to give about 90%
yield of Ar-ethyl-AP-[6-(4,4,5,5-tetrame^
LC/MS 3.5 min, 348 (M+l), 346 (M-l).
A 40 ml pressure tube was charged with about 1.5 ml ethylene glycol dimethyl ether, about
10 mg Pd(PPh3)4 and about 0.016 ml 2-bromo-5-chlorothiophene. The slurry was evacuated then
released to nitrogen atmosphere three times. Next 52 mgA^-ethyl-A^-[6-(4,4 5 5 s 5-tetramethyl-l ? 3,2-
dioxaborolan-2-yl)-l,3-benzothiazol-2-yl]urea was added and the slurry was again evacuated and
released to nitrogen three times. Following addition of sodium carbonate solution (about 46 mg in
about 0.5 ml water), the suspension was evacuated and released to nitrogen atmosphere 3 times. The
reaction tube was then sealed tightly and heated to about 85-90°C overnight. Upon cooling to room
temperature the now clear solution was purified by preparative HPLC to yield 43% of A^-[6-(5-
chloro-2-thienyl)-l,3-benzothiazol-2-yl]-A^-ethylurea. ] H NMR 1.1 (t, 3H), 3.2 (m, 2H), 6.75 (m,
1H), 7.18 (d, IH), 7.38 (m, 1H), 7.6 (m, 2H), 8.19 (d, 1H), 10.75 (br s, 1H); LC/MS 3.86 min, 338
(M+l), 336 (M-l).
Example 3 1 7
7V-EthyI-A^-[6-(177-l-pyrroIyl)-l,3-benzothiazol-2-yl]urea
Charged about 500 mg A^ethyl-A^-(6-nitro-l,3-benzothiazol-2-yl)urea into about 75 ml ethanol.
Added about 20 mg platinum oxide then evacuated and released to hydrogen three times. The system
was then put under hydrogen pressure (about 20-40 psi) for about 5-20 hours. The reaction was
stopped and the entire mass was fdtered through diatomaceous earth and washed with methanol. The
solvent was removed in vacuo and the crude ^-(e-amino-l^-benzothiazol^-yO-A^-ethylurea was
used for the next step without further purification. Charged about 0.44g 7^-(6-amino-l,3-
benzothiazol-2-yI)-A"-ethyIurea into about 15 ml acetic acid then added about 0.23 ml 2,5-
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dimethoxytetrahydrofuran. Refluxed for about 1 hour then cooled to room temperature. The solvent
was removed in vacuo and the product was purified by preparative HPLC. NMR 1.1 (t, 3H), 3.2
(m, 2H), 6.26 (m, 2H), 6.71 (m, 1H), 7.35 (m, 2H), 7.55 (m, 1H), 7.7 (m, 1H), 8.1 (m, 1H), 10.69 (br
s, 1H); LC/MS 3.1 min, 285 (M-I).
Example 3 1 8
(2-Amino-l,3-benzothiazol-6-yl)methyl cyanide
2 grams of 4-aminobenzonitriIe is dissolved in about 40mL acetic acid and the solution is cooled to
about 16°C. About 3.3g of potassium thiocyanate is added and the flask is equipped with an addition
funnel. The addition funnel is charged with about 2.7 g bromine and about 5 ml acetic acid. This
dark solution is then added to the benzonitrile solution in a dropwise fashion under good agitation
and allowed to stir for about 16 hours. The slurry is then drowned into water and filtered. The
presscake is washed well with water, reslurried in dilute aqueous alkali and filtered. Again the
presscake is washed well with water. After drying in vacuo, about 2 grams is isolated.
lH NMR 6.8 (d, 1H, J = 8.7 Hz), 6.9 (br s, 2H), 7.6 (dd, 1 H, J = 2 Hz, J = 8.7 Hz), 8.0 (d, 1H, J = 2
Hz), LC/MS 2.34 min, 174 (M-l), lab LC retention time 7.7 minutes.
Example 319
jV-(6-Cyano-l,3-benzothiazol-2-yl)-A^-ethylurea
0.2 grams of 2-amino-l,3-benzothiazoIe-6~carbonitrile is dissolved in about 5 ml
dimethylformamide. About 0.2 ml of ethylisocyanate is added followed by about 0.3 ml
triethylamine and the solution is heated to about 80°C under good agitation. The solution is allowed
to stir for about 4 hours then cooled to RT. The solvent is removed in vacuo and the solids are
washed well with ether. The product is further purified by column chromatography and after drying
in vacuo, about 0.14 grams is isolated.
lH NMR 1.1 (t,3H, J = 7.2 Hz), 3.2 (m, 2H), 6.8 (s, 1H), 7.7 (m, 2H), 8.4 (s, 1H), 11.0 (s, 1H),
LC/MS 2.54 min, 247 (M+l), 245 (M-l), lab LC retention time 7.8 minutes.
Example 320
7V-[6-(2-Aminoethyl)-l,3-benzothiazoI-2-yI]-A^-ethylurea
Charged about 500 mg A^(6-cyano-l,3-benzothiazol-2-yl)-A^-ethylurea into about 50 ml ethanol and
about 1 ml chloroform. Add about 0.1 grams platinum oxide then stir under 20-40 psi hydrogen for
about 8 hours. The solution is then basified with sodium bicarbonate to pH>7, filtered through a bed
of Celite® and washed well with ethyl acetate. The resulting crude product is purified by preparative
HPLC to yield unreacted N-(6-cyano-l,3-benzothiazol-2-yl)-A^-ethylurea and 7V-[6-(2-aminoethyl)-
1 ,3-benzothiazoi-2-y I]-A*-ethylurea (20%).
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iHNMR 1.08 (t, 3H), 2.7 (m, 2H), 2.9 (m, 2H), 3.16 (m 2H), 6.8 (br s, IH), 7.2 (m, 1H), 7.25 (br s 5
IH), 7.5 (m, IH), 7.95 (m, IH), LC/MS 2.09 min, 263 (M-l), 265 (M+l).
General Procedure for the reaction of A r -[6-(2-aminoethyl)-l,3-benzothiazol-2-yI1-7V T -ethylurea
5 with an isocyanate
0.02 grams of A r -[6-(2-aminoethyI)-l,3-benzothiazoI-2-yl]-iV l -ethylurea is dissolved in about 1 ml
dimethylformamide. About 2 equivalents of the appropriate isocyanate is added followed by about
0.02 ml triethylamine and the solution is heated to about 80°C under good agitation. The solution is
allowed to stir for about 10-24 hours then cooled to RT. The solvent is removed in vacuo and the
10 solids are washed well with ether. The product is further purified by preparative HPLC and dried in
vacuo.
Example 321
A r -[6-(ethylureido)methyl)-l,3-benzothiazol-2-yl]-A ?r, -ethylurea
LC/MS 2.65 min, 336 (M+l), 334 (M-l).
15 Example 322
A r -[6-(phenylureido)methyl)-l,3-benzothiazoI-2-yl]-A^ T -ethylurea
iHNMR 1.08 (m, 3H), 2.85 (m, 2H), 3.25 (m, 2H), 3.45 (m, 2H), 6.3 (m, IH), 6.75 (m, IH), 6.8-7.3
(m, 5H), 7.35 (m 5 IH), 7.45 (m, IH), 7.75 (s, IH), 8.43 (s, IH), 10.58 (br s, IH), LC/MS 3.4 min,
384 (M+l).
20 Example 323
iV-[6-(EthyI-2-amino-m-tolylurea)-l,3-benzothiazol-2-yl]-A^-ethyiurea
1 H NMR 1 .09 (m, 3H), 2.23 (s, 3H), 2.8 (m, 2H), 3. 1 7 (m, 2H), 3.35 (m, 2H), 6.07 (m, 1 H), 6.7 (m,
2H), 7.0-7.5 (m, 5H), 7.7 (s, IH), 8.35 (s, IH)", 10.57 (br s, IH), LC/MS 3.27 min, 398 (M+l).
Example 324
25 A^-(8-Cyano[l,3]thiazoIo[5%4 T :3,4]benzo[c]isoxazoI-2-yI)-A^-ethyIurea
0.2 g of [2-(3-ethyl-5-methyl-2-oxo- 1 ,3,5-triazinan- 1 -yl)-6-nitro- 1 ,3-benzothiazol-7-yl]methyl
cyanide was charged into about 2 ml dimethylformamide. About 15 equivalents of triethylamine
were then added followed by about 1 5 equivalents of trimethylsilyl chloride. The solution was stirred
at room temperature for about 4-20 hours. The reaction was quenched by pouring into dilute aqueous
30 hydrochloric acid then extracted well with ethyl acetate. The combined organics were back extracted
with dilute sodium bicarbonate then dried over magnesium sulfate and concentrated. The dark oil
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was further purified by preparative HPLC to yield 2-(3-ethyl-5-methyl-2-oxo-l,3,5-triazinan-l-
yl)[l ,3]thiazolo[5',4':3,4]benzo[c]isoxazol-8-yl cyanide.
lHNMR 1.12 (m, 3H), 2.55 (s, 3H), 3.4 (m, 2H), 4.39 (s, 2H), 5.16 (s, 2H), 7.9 (d, 1H), 7.95 (d,
1H), LC/MS 2.6 min, 343 (M+l).
5 The foregoing product was then deprotected with NHC1 in dioxane. Thus, the product was
dissolved in about 2 ml 4 NHC1 in dioxane. After stirring for about 2-8 hours, the crude reaction
mixture is poured onto ice and separated between water and ether. Following further extraction with
ether, the combined organic layers are dried with magnesium sulfate and concentrated. The product
is further purified by preparative HPLC.
10 lHNMRl.ll (t,3H), 3.22 (m, 2H), 6.8 (brs, 1H), 7.9 (d, 2H), 1 1.25 (br s, 1 H), LC/MS 2.24 min,
288 (M+l).
General procedure for preparation of ureas
0.2 grams of methyl [(6-cyano-l,3-benzothiazol-2-yl)amino]methanethioate is dissolved in about 5
ml of an alkanol. About 0.04 ml of pyridine is added followed by an excess of the appropriate amine
15 and the solution is heated to about 80°C under good agitation. The solution is allowed to stir for
about 14 hours then cooled to RT. The solvent is removed in vacuo. The product is further purified
by preparative HPLC then dried in vacuo. If necessary, maleate salts were prepared by dissolving in
an alkanol, then adding to this solution a solution of the appropriate amount of maleic acid in alkanol
solvent. Upon cooling, the maleate products were collected as a precipitate.
20 Example 325
A r -(6-Cyano-l,3-benzothiazol-2-yl)-A^-[3-(4-methylpiperazino)propyl]urea
lHNMR 1.6 (m, 2H), 2.1 (s, 3H), 2.3 (m, 2H), 2.7 (br s, 4H), 2.9 (br s, 4H), 3.1 (m, 2H), 7.65 (m,
1H), 7.95 (m, 2H), 8.35 (brs, 1H), 8.45 (brs, 1H), LC/MS 1.50 min, 359 (M+l).
Example 326
25 Ar-(6-Cyano-l^-benzothiazol-2-yl)-A^-(2-morpholinoethyI)urea
lHNMR 1.9 (s, 3H), 2.42 (m, 6H), 3.58 (m, 4H), 6.85 (br s, 1H), 7.73 (m, 2H), 8.43 (s, 1H), 9.8 (br
s, 1H), LC/MS 1.54 min, 332 (M+l).
Example 327
N-(6-Cyano-l,3-benzothiazol-2-yl)-A^-(3-(9-benzyl-9-azabicycIol3.3.1]nonyl))urea
30 1 H NMR 1 .48 (m, 2H), 1 .6-2.0 (m, 8H), 2.85 (br s, 2H), 3.82 (br s, 2H), 4.45 (m, 1 H), 6.7 (br s, 1 H),
7.22 (m, 1H), 7.35 (m, 4H), 7.75 (m, 2H), 8.46 (s, 1H), 10.9 (br s, 1H). LC/MS 2.16 min, 432
(M+l).
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Example 328
jy.(6-Cyano-13-benzothiazol-2-yl)-A^-[6-(4-methyIpiperazino)-3-pyridyl]urea
1HNMR 2.84 (s, 3H), 3.1 (br s, 4H), 3.49 (br s, 2H), 4.32 (br s, 2H), 6.125 (s, 2H), 6.99 (d, 1H), 7.8
(m,3H), 8.28 (s, 1H), 8.48 (s, 1H), 9.17 (s, 1H), 9.76 (br s, 1H). LC/MS 2.62 min, 394 (M+l), 392
5 (M-l).
Example 329
AT-(6-Cyano-l 5 3-benzothiazol-2-y])-A^-(3-(8-benzy]"8-azabicyclo{3.2.1]octyl))ur^
1HNMR 1.5-1.8 (m, 6H), 2.05 (m, 2H), 3.15 (m, 2H), 3.36 (br s, 2H), 3.95 (m, 1H), 6.7 (br s, 1H),
7.35 (m, 5H), 7.74 (m, 2H), 8.46 (s, 1H), 10.9 (br s, 1H). LC/MS 2.86 min, 418 (M+l), 416 (M-l).
10 Example 330
jV-(6-Cyano-lv3-benzothiazol^
iHNMR 1.3-1.6 (m, 6H), 1.8 (br s, 1H), 1.9 (br s, 2H), 3.1 (m, 2H), 3.2 (m, 2H), 3.5 (br s, 2H), 6.8
(br s, 1H), 7.35 (m, 5H), 7.74 (m, 2H), 8.45 (s, 1H), 1 1.8 (br s, 1H). LC/MS 3.09 min, 432 (M+l),
430 (M-l).
15 Example 33 1
tert-Butyl 4-[([(6-cyano-l,3-benzothiazol-2-yl)amino]carbonylamino)methyl]-l-
piperidinecarboxylate
iHNMR 1.1 (m, 2H), 1.38 (s, 9H), 1.62 (m, 3H), 2.7 (m, 2H), 3.1 (m, 2H), 3.95 (m, 2H), 6.9 (br s,
1H), 7.74 (m, 2H), 8.45 (s, 1H), 11.0(brs, 1H). LC/MS 2.61 min, 414 (M-l).
20 Example 332
A r -(6-Cyano-l,3-benzothiazol-2-yI)-A^-(4-piperidylmethyl)urea
iHNMR 1.1 (m, 2H), 1.6 (m,3H),3.1 (m,4H) 5 3.4 (m 5 4H), 7.32 (br s, 1H),7.6 (d, 1H),7.67 (d,
1H), 7.95 (s, 1H), 8.31 (br s, 1H). LC/MS 1.54 min, 316 (M+l).
Example 333
25 /er/-Butyl 4-[2-([(6-cyano-l,3-benzothiazol-2-yl)amino]carbonylamino)ethyl]-l-
piperazinecarboxylate
iHNMR 1.4 (s, 9H), 1.9 (s, 3H), 2.4 (m, 6H), 3.3 (m, 4H), 6.85 (br s, 1H), 7.74 (m, 2H), 8.45 (s,
1H), 11.2 (brs, 1H), other signals under DMSO or water peaks. LC/MS 2.81 min, 431 (M+l X 429
(M-l).
30 Example 334
A r -(6-Cyano-l,3-benzothiazol-2-yI)-A^-(2-piperazinoethyl)urea
iHNMR 1.9 (s, 6H), 2.4 (m, 6H), 2.75 (m, 4H), 3.3 (m, 2H), 3.5 (br s, 1H), 7.15 (br s, IH), 7.7 (m,
2H), 7.95 (s, 1H), 8.4 (s, 1H). LC/MS 2.58 min, 331(M+1), 329 (M-l).
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Example 335
A^(6-Cyano-l^-benzothiazol-2-yl)-A^-[4-(4-methylpiperazino)cyclohexyl]urea More polar
(trans)
lHNMR 1.2 (m, 4H), 1.8 (m, 2H), 1.91 (s, 3H), 1.93 (m, 2H), 2.33 (m, overlapping with DMSO),
5 3.36 (m, overlapping with water), 6.7 (m, 1H), 7.7 (m, 2H), 8.45 (s, 1H), 10.8 (m, 1H), other signals
under DMSO or water. LC/MS 1 .64 min, 399 (M+l ).
Example 336
Ar-(6-Cyano-W-ben^ Less P olar < cis )
1 H NMR 1 .55- 1 .75 (m, 4H), 2.2 (m, overlapping with DMSO), 3 .85 (m, 1 H), 6.95 (m, 1 H), 7.7 (m,
10 2H), 8.46 (s, 1H), 10.65 (m, 1H), other signals under DMSO or water. LC/MS 1.75 min, 399 (M+l).
Example 337
^.(g-Cyano-l^-benzothiazol^-yO-TV-CS-piperidinopropyOurea
lHNMR 1.35 (m, 2H), 1-6 (m, 4H), 1.75 (m, 2H), 1.9 (s, 3H), 2.3 (m, 6H), 3.25 (m, 2H), 6.9 (m,
1H), 7.7 (m, 2H), 8.4 (s, 1H), 10.8 (brs, 1H), LC/MS 1.69 min, 344 (M+l).
15 Example 338
2-[(Ethylamino)carbonyl]amino-l^-benzothiazole-6-carboxylic acid
About 60 mg A^-(6-cyano-l,3-benzothiazol-2-yl)-A^-ethylurea was charged into about 5 mL of about
a 1:1 mixture of about 2N aq KOH and dioxane. The reaction mixture was then brought to reflux for
about 12-24 hours. Upon cooling, the reaction mixture was poured into about 25 mL of dilute
20 aqueous acid. The white precipitate was collected by filtration and was washed well with water. *H
NMR 1.09 (t, 3H), 3.19 (m, 2H), 6.79 (br s, 1H), 7.65 (d, 1H), 7.92 (m, 1H), 8.48 (d, 1H), 1 1.0 (br s 5
1H) 5 12.8 (brs, 1H); LC/MS 2.12 min, 266 (M+l ) 5 264 (M-l), lab LC retention time 4.7 minutes. •
Example 339
iV-(6-Bromo-l,3-benzothiazol-2-yl)-7V-ethylurea
25 About 5 g of 4-bromoaniiine is dissolved in about lOOmL acetic acid and the solution is cooled to
about 16 °C. About 5.6 g of potassiumthiocyanate is added and the flask is equipped with an
addition funnel. The addition funnel is charged with about 4.7 g bromine and about 20 mL acetic
acid. This dark solution is then added to the benzonitrile solution in a dropwise fashion under good
agitation and allowed to stir for about 6-20 hours. The slurry is then drowned into water and filtered.
30 The presscake is washed well with water, dilute alkali, and then water and filtered. LC/MS confirms
product is a mixture of 6-bromo-l,3-benzothiazol-2-amine and 2-amino-l>3-benzothiazol-6-yl
thiocyanate which was carried on to the next step without further purification.
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About 0.75 g of 6-bromo-l,3-benzothiazol-2-amine (crude) is dissolved in about 15 mL
DMF. About 0.5 mL of ethyl isocyanate is added followed by about 0.9 mL triethylamine and the
solution is heated to about 80 °C under good agitation. The solution is allowed to stir for about 4
hours then cooled to RT. The solvent is removed in vacuo and the solids are washed well with ether.
The product is further purified by column chromatography.
lHNMR 1.08 (t, 3H), 3.19 (m, 2H), 6J\ (s, 1H), 7.48 (m, 1H), 7.54 (d, 1H), 8.13 (d, 1H), 10.75 (br
s, 1H); LC/MS 3.78 min, 301 (M+l), lab LC retention time 8.9 minutes.
Example 340
2-(((Ethylamino)carbonyI)amino)-l^-benzothiazol-6-yl thiocyanate
Also islated the title compound from the reaction mixture of Example ^HNMR 1.09 (t, 3H), 3.19
(m, 2H), 6.75 (s, 1H), 7.62 (m, 1H), 7.71 (d, 1H), 8.3 (d, 1H), 10.9 (br s, 1H); LC/MS 3.0 min, 279
(M+l), lab LC retention time 7.8 minutes.
Example 341
2-[(Ethylainino)carbonyl]amino-l ? 3-benzothiazole-6-carboxamide
About 1 g of AT-(6-cyano-l 9 3-benzothiazol-2-yI)-A' l -ethylurea was charged into about 30 mL of
aqueous alkanol. Added about 0.6 g hydroxylamine hydrochloride and about 0.45 g sodium
carbonate then heated to reflux. The solution was refluxed for about 4-8 hours then cooled to room
temperature. The precipitate was recovered by filtration and washed well with water. LC/MS
indicated a mixture of two products identified as the following after further purification by
preparative HPLC:
lHNMRl.09 (t, 3H),3.16(m, 2H), 5.8 (s, 2H),7.1 (brs, 1H), 7.55 (d 5 1H), 7.67 (m, 1H),8.1 (d,
1H); LC/MS 2.1 min, 265 (M+l), 263 (M-l).
2-[(Ethylamino)carbonyl]amino-l,3-benzothiazole-6-carboxamideoxime
Also isolated this title product from the reaction mixture of Example ^HNMR 1.09 (t ? 3H), 3.19 (m,
5 2H),5.81 (br s, 2H), 6.72 (br s, lH),7.57(d 5 lH),7.68(m, lH),8.13(d, lH),9.58(s, 1H), 10.7 (br
s, 1H); LC/MS 2.04 min, 278 (M-l).
Example 342
A^-Ethyl-iV-[6-(5«methyl-l,2,4«oxadiazol-3-yl)-l^-benzothiazoI-2-yl]urea
About 50 mg of 2-[(ethylamino)carbonyl]amino-l,3-benzothiazole-6-carboxamideoxime was
0 charged into about 1 mL of glacial acetic acid. The mixture was heated to about 1 10 °C then stirred
at this temperature for about 12-20 hours. The solution was cooled to room temperature and the
solvent was removed under reduced pressure. The residue was then further purified by preparative
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HPLC. 1HNMR 1.10 (t, 3H), 2.67' (s, 3H), 3.17 (m, 2H), 6.74 (s, 1H), 7.73 (d, 1H), 7.97 (d, 1H),
8.53 (s, 1H), 10.9 (s, 1H); LC/MS 2.69 min. 304 (M+l), 302 (M-l).
Example 343
AT-(6-Anilino-l^-benzothiazol-2-yl)-iV-ethylurea
5 About 150 mg of Ar-(6-amino-l,3-benzothiazol-2-yl)-/V'-ethylurea, and about 285 mg triphenyl
bismuthane were charged into about 15 mL dichloromethane. Introduced about 0.1 mL triethylamine
and then about 120 mg of copper(Il) acetate. The mixture was stirred at RT for about 12-24 hours.
The solution was poured into about 60 mL of dilute aqueous acid then stirred for about 1 hour at
room temperature. The crude mixture was extracted with dichloromethane and the combined
10 organics were washed with dilute aqueous acid, then water, then dilute aqueous potassium carbonate
then dried. The solvent was removed under reduced pressure. The crude mixture was further purified
by preparative HPLC. iHNMR 1.09 (t, 3H), 3.18 (m, 2H), 6.69 (s, 1H), 6.78 (m, 2H), 7.04 (d, 2H),
7.09 (m, 1H), 7.21 (m, 2H), 7.49 (d, 1H), 7.55 (d, 1H), 8.13 (s, 1H), 10.46 (br s, 1H); LC/MS 3.06
min, 313 (M+l), 311 (M-l).
, 5 Example 344
iV-[6-(Aminomethyl)-l^-benzothiazol-2-yll-A^-ethylurea
About 0.05 g of iV-(6-cyano-l,3-benzothiazol-2-yl)-iV-ethylurea was charged into about 10 mL of
ethyleneglycol dimethylether. Introduced about 50 mg lithium aluminum hydride in several portions
over about 12-24 hours. The slurry was quenched with about 5-10 mL ethyl acetate then about 1-2
20 mL of saturated aqueous sodium sulfate. The slurry was then diluted with water and extracted with
ethyl acetate. Following drying and removal of solvents under reduced pressure, the product was
isolated by preparative HPLC. lH NMR 1.08 (t, 3H), 1.88 (s, 3H), 3.17 (m, 2H), 3.83 (s, 2H), 7.07
(m, 1H), 7.32 (m, 1H), 7.54 (d, 1H), 7.8 (s. 1H); LC/MS 1.86 min, 251 (M+l), 249 (M-l).
Example 345
25 iV.[6-(Ethylureido)methyl)-l,3-benzothiazol-2-yl]-/r-ethylurea
About 0.025 g of 7V-[6-(aminomethyl)-l,3-benzothiazol-2-yl]-/V-ethylurea was dissolved in about 1
mL dimethylformamide. About 0.015 mL of ethylisocyanate was added followed by about 0.029 mL
triethylamine and the solution was heated to about 80 °C under good agitation. The solution was
allowed to stir for about 4 hours then cooled to RT. The solvent was removed in vacuo and the solids
30 were washed well with ether. ^H NMR 1.0 (t, 3H), 1.09 (t, 3H), 3.0 (m, 2H), 3.2 (m, 2H), 4.25 (d,
2H), 5.86 (m, 1H), 5.33 (br s, 1H), 6.3 (m, 1H), 6.7 (m, 1H), 7.23 (m, 1H), 7.53 (d, 1H), 7.69 (d,
1H), 10.6 (br s, 1H); LC/MS 1.43 min, 322 (M+l).
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Example 346
AMEthyl-A^6-(2Z^l,2rM^
About 30 mg A^-(6-cyano-l,3-benzothiazol-2-yl)-A^-ethylurea was charged into about 5 mL dry
tetrahydrofuran. Added about 2 g of azidotributylstannane then refluxed for about 48-72 hours. The
solvent was removed under reduced pressure then the crude oil was taken up in dichloromethane.
About 0.5 mL of dilute aqueous hydrochloric acid was added and a white precipitate formed. This
was allowed to sit for about 30 minutes then the precipitate was collected by filtration, washed with
warm THF then dried under vacuum. lH NMR 1.10 (t, 3H), 3.2 (m, 2H), 6.76 (br s, 1H), 7.78 (d,
1H), 8.0 (d, 1H), 8.58 (s 5 1H), 10.91 (s, 1H); LC/MS 1.37 min, 290 (M+l).
Example 347
M-[(2-[(Ethylamino)carbonyl]^
About 0.015 g of ^-[e-CaminomethyO-l^-benzothiazol^-ylj-TV-ethylurea is dissolved into about 1
mL dichloromethane. Cooled to about 0-5°C then added about 0.01 mL of triethylamine followed by
about 1.2 equivalents of the appropriate sulfonylchloride. The reaction is warmed to room
temperature then stirred for about 12-24 hours. The solvent is removed under reduced pressure and
the reaction crude is further pruified by preparative HPLC. NMR 1.08 (t, 3H), 3.19 (m, 2H), 4.05
(d, 2H), 6,71 (br s, 1H), 7.20 (m 5 1H), 7.5 (d, 1H), 7.54-7.65 (m, 4H), 7.8 (d, 1H), 8.16 (m, 1H), 10.6
(br s, 1H); LC/MS 1.98 min, 391 (M+l).
Example 348
^-[(2-[(Ethylamino)carbonyl]amino-l,3-benzothiazol-6-
yI)methyI]trifluoromethanesu!fonamide
Also isolated the title compound from the reaction mixture of Example 347 *HNMR 1.09 (t, 3H),
3.18 (m, 2H),4.4 (s, 2H),6.71 (m, 1H), 6.6 (d, 1H), 7.32 (m, 1H),7.81 (s, 1H),9.93 (br s, 1H),
10.68 (br s, 1H); LC/MS 2.15 min, 383 (M+l).
Example 349
A^6-Phenyl-2-[(ethylamino)carbonyl]amino-l,3-benzothiazole-6-carboxamide
About 0.2 g of 2-[(ethyIamino)carbonyl]amino-l,3-benzothiazole-6-carboxyIic acid was dissolved
into about 20 mL dichloromethane and about 0.2 mL triethylamine. Added about 1 eq of the
appropriate amine followed by about 0.3 g of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride then stirred at room temperature for about 12-24 hours. The solvent was removed
under reduced pressure then the crude reaction mixture was purified by preparative HPLC. NMR
1.10 (t, 3H) 5 3.2 (m, 2H), 6.76 (br s, 1H), 7.10 (m, 1H), 7.36 (m, 2H), 7.70 (d, 1H), 7.79 (d, 2H),
7.97 (m, 1H), 8.5 (s, 1H), 10.23 (s, 1H), 10.9(brs, 1H); LC/MS 2.86 min, 341 (M+l);
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Example 350
7V6-[3-(4-MethyIpiperazino)pro
carboxamide
Also isolated the title compound from the reaction mixture of Example 1 H MMR 1 .09 (t, 3H), 1 .76
5 (m, 2H), 2.6-2.7 (m, 4H), 3.2 (m, 4H), 3.3 (m, 4H), 3.42 (m, 2H), 6.12 (s, 4H), 6.75 (m, 1H), 7.64 (d,
1H), 7.84 (m, 1H), 8.34 (d, 1H), 8.49 (m, 1H), 10.9 (br s, 1H), one methyl group under solvent
peaks; LC/MS 1.34 min, 405 (M+l).
General procedure for preparation of ureas
0.2 grams of methyl [(6-cyano-l ? 3-benzothiazol-2-yl)amino]methanethioate is dissolved in about 5
10 ml of an alkanol. About 0.04 ml of pyridine is added followed by an excess of the appropriate amine
and the solution is heated to about 80 °C under good agitation. The solution is allowed to stir for
about 14 hours then cooled to RT. The solvent is removed in vacuo. The product is further purified
by preparative HPLC then dried in vacuo. If necessary, maleate salts were prepared by dissolving in
an alkanol, then adding to it a solution of the appropriate amount of maleic acid in the same alkanol
15 solvent. Upon cooling, the maleate products were collected as a precipitate.
Example 35 1
7V-(6-Cyano-l^-benzothiazoU2-yl)-iV , -[6-(4-niethyIpiperazino)-3-pyridyI]urea
lHNMR 2.84 (s, 3H), 3.1 (br s, 4H), 3.49 (br s, 2H), 4.32 (br s, 2H), 6.125 (s, 2H), 6.99 (d, 1H), 7.8
(m, 3H), 8.28 (s, 1H), 8.48 (s, 1H), 9.17 (s, 1H), 9.76 (br s, 1H). LC/MS 2,62 min, 394 (M+l), 392
20 (M-l).
Example 352
^-(e-Methylcyano-l^-benzothiazol-I-yO^TV-ethylurea
About 1 g of (2-amino-l,3-benzothiazol-6-yl)methyl cyanide is dissolved in about 10 ml
dimethylformamide. About 0,8 ml of ethylisocyanate is added followed by about 1.4 ml
25 triethylamine and the solution is heated to about 80 °C under good agitation. The solution is allowed
to stir for about 4-6 hours then cooled to RT. The solvent is removed in vacuo and the solids are
washed well with ether. The product is recrystallized from hot EtOAc. *H NMR 1 .09 (t, 3H), 3.1 (m,
2H), 4.1 (s, 2H), 6.72 (m, 1H), 7.32 (m, 1H), 7.61 (d, 1H), 7.85 (s, 1H), 10.7 (s, 1H); LC/MS 2.73
min, 261 (M+l), 259 (M-l).
30
Example 353
Ar-[6-(Di[(5-methyI-2-fui^
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15
20
About 10 mg of A^(6-aminomethyl-l,3-benzothiazol-2-yl)-iV , -ethyIurea was dissolved in about 1
mL dichloromethane, 3 jliL acetic acid, and 4 \xh 5-methyl-2furfural. Stirred for about 1 hour at room
temperature then about 0.013 g of sodiumtriacetoxyborohydride was added. The mixture was stirred
at room teperature for about 12-20 hours. The reaction was drowned into about 5 mL water then
extracted well with dichloromethane. The combined organics were dried over magnesium sulfate
and solvent was removed under reduced pressure. The crude mixture was further purified by
preparative HPLC. ] H NMR 1.10 (t 5 3H), 1.8 (s, 3H), 2.2 (s, 6H), 3.1 (m, 2H), 3.5 (s, 4H), 3.6 (s,
2H), 6.0 (m, 2H), 6.2 (m, 2H), 7.29 (m, 1H), 7.4 (br s s 1H), 7.5 (m, 1H), 7.7 (s, 1H); LC/MS 2.87
min, 439 (M+l).
General procedure for making compounds of the formula n 1-1 starting
from a substituted aniline.
To a stirring solution of a substituted aniline, such as 4-(p-flourophenylthio)aniline and
potassium thiocyanate (-2 eq.) in glacial acetic acid at room temperature was added dropwise a
solution of bromine (-1 eq.) in glacial acetic acid. The reaction mixture was stirred for about 24
hours. The solid (KBr) was removed by filtration and the resulting solution concentrated in vacuo to
give the corresponding benzothiazolyl as the HBr salt. HPLC RT = 3.3 min, MH+ 277.
To a stirring solution of the benzothiazolyl from the preceding step and triethylamine (~2
eq.) in toluene was added ethyl isocyanate (-1 .5 eq.). The reaction mixture was heated to about 80
degrees for about 2 days. The precipitated product was collected on a fritted funnel, washed with
diethyl ether and dried in vacuo. HPLC RT - 3.61min, MH+ 349.
The following examples were synthesized according to the procedures described
hereinabove.
25
Ex. #
W
R'
R 3
HPLC
(min.)
MH"
354
-CH 2 -OH
H
ethyl
2.22
434
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355
~CH 2 -0-C(0)->JH-Et
H
etnyi
O /l*7
Z.4 /
*^H7
JU /
356
/~i i t
-S-phenyl
H
etnyi
Z.ZZ
357
-O-phenyl
H
etnyl
Z. /U
358
-S-CH 3
H
etnyi
J O J
359
-C(0)-phenyI
H
etnyi
J.oO
360
-S(0)-phenyl
H
etnyl
J) . /-)
J5 77
361
-S-p-nitrophenyl
T T
H
etnyi
t
362
-S-p-methylphenyl
H
ethyl
J . 1
-S-/?-chlorophenyl
CN
etnyl
1 Id
.5 .zu
364
-S-p-methoxyphenyl
CN
ethyl
2.77
368
365
-S-w-CF 3 -phenyl
CN
ethyl
z.v /
HA 1
366
-S-o-chlorophenyl
H
ethyl
AA 1
367
-C(0)-CH 3
H
ethyl
3.40
441
368
-NH-C(0)-NH-(-CH,) 2 -2-
thienyl
H
ethyl
1.71
352
369
-NH-C(0)-NH-3-pyridyl
H
ethyl
1.91
A "\ A
414
370
-S(0) 2 -p-
(carboxymethylamino)-
phenyl
H
ethyl
j>.l j
40j>
371
-N-morphoIino
H
ethyl
\> J>
372
-NH-C(0)-NH-Et
H
ethyl
J . JZ
-NH-C(0)-NH-CH 2 — phenyl
H
ethyl
1.81
425
374
S-/?-chlorophenyl
H
ethyl
2.29
252
375
-S-p-bromophenyl
H
ethyl
2.65
323
376
-S-m-CF 3 -phenyi
H
ethyl
3.53
330
377
no 2
H
A
Ph
3.29
314
378
NO,
H
3.02
268
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379
NO,
H
3.44
255
380
CI
H
-(CH 2 ) 3 -4-methylpiperazin- 1 -yl
2.53
346
381
CI
H
3.56
375
■382
CI
H
r" ph
344
383
CI
H
Ph
3.72
389
384
NO,
H
-(CH 2 ) 2 -N-morpholino
-XJ /
385
385
NO,
H
(' x > — N N— Me
3.80
423
*
386
CI
H
(/ x ) — N N— Me
\=/ \—J
3.69
364
387
CI
H
-(CH 2 ) 2 -N-morpholino
1.79
264
389
CI
H
0
/ \ II
2.72
390
390
CI
H
-CH 2 -piperidin-4-yl
2.30
357
391
-S-/?-fluorophenyl
H
-(CH 2 )3-4-methylpiperazin- 1 -yl
2.32
461
Ex#
W
R 1
R 2
HPLC (min)
MPT
392
-OCF 3
H
H
2.43
306
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393
-OEt
H
H
2.12
266
394
F
H
H
2.03
240
395
H
H
4-C1
2.30
256
396
H
H
4-CH3
2.25
236
397
CH 3
H
H
2.17
236
398
CH,
H
5-CH3
2.71
276
399
-OCH 3
H
H
2.37
250
400
-S0 2 -Me
H
H
K95
252
401
NH 2
H
H
1,75
300
402
-NH~C(0)-Me
H
H
2.51
237
403
-NH-CH.-phenyl
H
H
2.58
279
404
H
H
5-CH3
3.28
327
405
H
F
5-F
3.12
266
406
H
H
5-C1
3.21
258
407
-NH-S(0)n-2-thienyl
H
H
3.38
256
408
-NH-S(OW3,5-
dimethylisoxazol-4-yI)
H
H
3,17
383
409
-NH-S(0) r Me
H
H
3,10
396
410
-NH-S(0) 2 -CH r phenyl
H
H
2.81
315
41 1
-N H-C(0)-0-CH 2 -CCI 3
H
H
2.77
391
412
-NH-C(0)-0-CH r Ph
H
H
2.16
412
413
-NH-C(0)-0-Me
H
H
3.47
371
414
NO,
H
4-CH3
2,87
295
415
-CH 2 -S(0) r phenyl
H
3.22
281
416
-OCH 3
H
5-OCH3
2.56
282
5
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CLAIMS
What is claimed is:
10
15
20
A compound of formula (I)
R 1
(I),
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein,
Q is H or represents a bond which is taken together with X 1 and the two nitrogen atoms to which Q
and X 1 are attached and the C=Y group to which the two nitrogen atoms are attached to form
Y
^ N
N — R'
N"
I,
Q
Ql is (C]-C6)alkyl;
Y is O or S;
W is H, CI, Br, I, N0 2 , CN, SCN, OCF 3 , -X q -(C(RlO) 2 ) a -Yl q -(C(R 10 )2)a-Z 1 q > °r an optionally
substituted group selected from the group consisting of alkyl, alkenyl, alkynyl, heterocyclyl-alkenyl,
and heterocyclyl-alkynyl;
Y ' and X are each independently selected from the group consisting of phenyl, heterocyclyl,
NRlO, O, S, SO, S0 2 , CF 2 , CFR, C=0, (C=0)NR 10 , SONRlO, SO2NRIO, NRl0(C=O),
nrIOso,
R
I
.N
10
NR'0sO 2j NR10SO2NR 10 ,NR10(C=O)NR10, 0 , 0
o
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10 D 10
I I I
O
o , o °
and
q for each occurrence is independently 0 or 1 ;
a for each occurrence is independently 0 or an integer from 1 to 5;
R.10 for each occurrence is independently selected from the group consisting of H,
optionally substituted aryl, optionally substituted heterocyclyl andan optionally substituted
alkyl group optionally substituted with one or more of the following: a C]_6 alkyl group
optionally substituted by one or more hydroxy, halo or optionally substituted amino; a Ci_6
alkoxy group optionally substituted by one or more hydroxy, halo or optionally substituted
amino; hydroxy; halo; or optionally substituted amino;
Zl is H, optionally substituted alkyl, optionally substituted aryl or optionally substituted
heterocyclyl;
Xl is hydrogen, alkyl, hydroxyalkyl or represents a bond which is taken together with R 3 as
described below or represents a bond which is taken together with Q as described above;
Rl and R 2 are each independently hydrogen, halogen, hydroxy, nitro, cyano, COOH, COOX 3 , SX 3 ,
S0 2 X 3 , SOX 3 , C(0)X 3 , NHC(0)X 3 , C(0)NHX 3 , NHS0 2 X 3 or selected from an optionally
substituted group consisting of alkyl, alkenyl, alkynyl, alkoxy, amino, NHX 3 , NX 3 X 3 , alkylamino,
arylamino, heterocyclylamino, alkylthio, alkylsulfonato, aryl, aryloxy, arylalkyl, arylalkenyl,
arylalkynyl, arylalkyloxy, heterocyclyl, heterocyclyloxy, heterocyclyl-alkyl, heterocyclyl-alkenyl,
heterocyclyl-alkynyl, heterocyclyl-alkylbxy, heterocyclylthio, heterocyclylsulfinyl,
heterocyclylsulfonyl, cycloalkyl, -(CH 2 ) m -(CHX2)CN, -(CH 2 ) m -(CHX2)COOH, -(CH 2 ) m -
(CHX2)COOX 3 , -(CH 2 ) m -(CHX2)S0 2 X 3 , -(CH 2 ) m -(CHX2)C(0)X 3 , -<CH 2 ) m -
(CHX 2 )C(0)NHX 3 and
-(CH 2 ) m -(CHX2)NHS0 2 X 3 ;
where m is 0 to 4;
X2 for each occurrence is independently H or an optionally substituted moiety selected from
the group consisting of alkyl, alkenyl, alkynyl, carbonyl, S(0) p alkyl, S(0) p aryi,
S(0) p heterocyclyl, amino, alkoxy, alkylthio, arylthio, perhaloalkyl, aryl, aryloxy, arylalkyl,
arylalkyloxy, heterocyclyl and heterocyclyl-alkyl;
p is 0, 1 or 2;
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X 3 for each occurrence is independently H or an optionally substituted moiety selected from
the group consisting of mono- or di-alkylamino, alkyl, alkenyl, alkynyl, aryl, arylalkyl,
heterocyclyl and heterocyclyl-alkyl;
or when R 1 is in the 7-position of the benzothiazole ring, R 1 and W can be taken together with the
5 carbon atoms to which they are attached to form an optionally substituted 5- or 6-membered
heterocyclyl ring;
R 3 is hydrogen, or an optionally substituted moiety selected from the group consisting of carbonyl,
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heterocyclyl, heterocyclyl-alkyl, heterocyclyl-
heterocyclyl, heterocyclyl-cycloalkyl, amino, alkylamino, arylamino, alkoxy, thioalkoxy and acyl;
10 or R 3 and X 1 are taken together with the nitrogen atom to which they are attached to form
-N' - <Z) "
or
O
where Z for each occurrence is independently selected from the group consisting of oxo, or
an optionally substituted moiety selected from the group consisting of-C(0)(Ci -Chalky],
-C(0)ary 1, -C(0)N(C i ~C 6 )alky 1, -C(0)N-ary 1, (C j -C 6 )alkyl, (C 2 -C 6 )alkenyl, (C 2 -
15 C6)alkynyl, amino, mono- or di-(Ci~C 6 )alkylamino 5 -COO(Ci-C6)alkyl, pyridyl, phenyl,
phenyl(C]-C6)alkyl and phenyl(C]-C6)alkenyl;
w h ere eac h of the optionally substituted moieties described hereinabove is optionally substituted by
one or more substituents each independently selected from the group consisting of oxo, amino, nitro,
mono- or bi-(C]-C6)alkylamino 5 hydroxy, nitrile, chloro, fluoro, bromo, iodo, CF3, (C]-C6)alkyl,
20 -C(0)(Ci-C 6 )alkyl, -COOH, -COO(Ci-C 6 )alkyl, -S-(Ci-C 6 )alkyI, ^S-aryl, (C]-C 6 )alkoxy, -
S0 2 NH 2 , phenyl, phenyl(Ci-C 6 )alkyl, -0-(Ci-C 6 )aIkyl-OH, -0-(Ci-C 6 )alkyl-0-(Ci-C 6 )alkyl, -O-
(C 2 -C 6 )alky 1-N-((C 1 -C 6 )alkyl) n , -N-(C 1 -C 6 )alky 1-OH, -N-(C 1 -C 6 )alkyl-0-(C 1 -C 6 )alky 1, -
C(0)NH 25 -C(0)N((Ci-C 6 )alkyl) n , ,-S(0) n (Ci-C 6 )alkyl, -S(0) n aryl -S(0) n heterocyclyl, and
heterocyclyl, where the alkyl groups mentioned herein optionally have one or more unsaturated
25 bonds in the alkyl portion;
n is 0, 1 or 2;
provided that
1) when Q is H; Y is O; R 1 and R 2 are each hydrogen, halogen, alkyl, alkoxy, alkylthio,
carboxyalkyl or optionally substituted phenyl; and X 1 is hydrogen or alkyl; then R 3 is not alkyl,
30 alkenyl, alkoxy, cycloalkyl or optionally substituted phenyl;
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2) when Q is H; Y is O; R 1 and R 2 are each hydrogen, halogen, alkyl, alkoxy, alkylthio,
carboxyalkyl or optionally substituted phenyl; then X 1 and R 3 are not taken together to form
■(Z)n
or L
3) when W is CI, Br or I; Q is hydrogen; Y is O; X 1 is H; then R 3 is not
5 optionally substituted by 1 to 3 substituents independently selected from the group consisting of
amino, mono- or bi-(C j-C6)alkylamino, hydroxy, chloro, fluoro, bromo, iodo, (Ci-C6)alkyl, (Cj-
C6)alkoxy and -S0 2 NH 2 ;
4) when W is CI, Br or I; Q is H; R 1 is 7-C1; R 2 is H; and X* is alkyl; then R 3 is not alkyl, alkoxy or
cycloalkyl;
10 5) when W is CI, Br or I; Q is H; R ] is 7-C1; R 2 is H; and X 1 is H; then R 3 is not alkyl or
cycloalkylamino;
6) when W is CI, Br, I or N0 2 ; Q is H; Y is O; X^ is H; Rl is OH; R 2 is N0 2 , amino, alkyl, alkoxy,
hydroxy lower alkyl or dialkylamino; then R 3 is not H or alkyl;
7) when W is CI, Br or I; Q is H; Y is O; R 1 is CF 3 , CH 2 F, NOo, alkyl or alkoxy; R 2 is H; X* is H;
15 then R 3 is not naphthyl or phenyl optionally substituted with halo, CF3, alkyl or alkoxy;
8) when W is CI, Br or I; Q is H; R 1 is alkyl; R 2 is H; X 1 is H or alkyl; then R 3 is not alkyl or
alkoxy;
9) when W is CI; Q is H; Y is S; R 1 and R 2 are each H; X 1 is H; then R 3 is not ethyl;
1 0) when W is CI; Q is H; Y is O; R 1 and R 2 are each H; X 1 is H; then R 3 is not n-butyl; and
20 11) when W is H, then R 1 and R 2 are not H at the same time.
2. A compound according to claim 1, racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein
the alkyl, alkenyl and alkynyl moieties, and the alkyl portion of a moiety is an optionally substituted
25 straight or branched chain having one to eight carbon atoms;
the aryl moiety and the aryl portion of a moiety is an optionally substituted phenyl,
naphthyl;
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the heterocyclyl moiety and the heterocyclyl portion of a moiety are selected from the group
consisting of an optionally substituted piperidinyl, pyridyl, pyrazinyl, pyrimidinyl, thienyl,
pyrrolidinyl, piperazinyl, thiomorpholinyl, morpholinyl, 2,3,4,5-tetrahydrofuranyl, 1,3-dioxanyl, 1,4-
dioxanyl, furanyl, and 1,2,4-triazolyl, tetrazolyU imidazolyl, pyrazolyl, thiazolyl, oxazolyl,
oxadiazolyl, thiadiazolyl, benzimidazolyL 1,3-dioxolanyl, 2-imidazolinyl, imidazolidinyl, 2-
pyrazolinyl, pyrazolidinyl, isothiazolyl, 1,2,3-triazolyl, 2H-pyranyl, 4H-pyranyl, 1,4-dithianyl, 1,3,5-
triazinyl, 1,3,5-trithianyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, purinyl, 4H-quinolizinyl,
cinnolinyl, phthalazinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, 1,8-naphthpyridinyl,
pteridinyl, quinuclidinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, pyrrolyl,
isoxazolyl, pyridazinyl, indazolyl, benzoxazolyl, benzofuranyi, benzothiazolyl, indolizinyl,
imidazopyridinyl and benzothienyh
3. A compound according to claim 2, racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutical ly-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein
r3 i s an optionally substituted moiety selected from the group consisting of (Ci-C8)alkyl, phenyl,
phenyl(Ci-C8)alkyl, thienyl, thienyl(Ci-C8)alkyl, piperidinyl, piperidinyl(Ci-C8)alkyl,
pyrrolidinyl, pyrrolidinyl(Ci-C8)alkyl, morpholinyl, morphoHnyl(C]-C8)alkyl, 2,3,4,5-
tetrahydrofuranyl, 2,3,4 5 5-tetrahydrofuranyl(Ci-C 8 )alkyl, furanyl, furanyl (C]-C 8 )alkyl, cycloalkyl,
cycloalkyl(Ci-C 8 )alkyl, pyridyl, pyridyl(Ci-C 8 )alkyl, 1,2,4-triazolyl, l,2,4-triazolyl(Ci-C 8 )alkyl,
4.
O »(C r C 8 )
and
A compound of formula (IA),
,1
N — R*
(IA),
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharmaceutical ly-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein
W is NO2 or CN;
Y is O or S;
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Rl is in the 7-position and is hydrogen, methyl, ethyl, allyl, phenyl, benzyl, -CH2-C(0)-CH3, -CH2-
C02-t-Bu, -CH2-S02-aryl, -alkyl-CN, or -aIkyI(CN)(CH2-aryl);
X* is hydrogen, alkyl or hydroxyalkyl;
r3 i s selected from the group consisting of ethyl, n-butyl, t-butyl, n-propyl, allyl, hydroxyalkyl,
5 aminoalkyl, -alkyl-NH-alkyl-OH, -alkyl-0-alkyl-OH 5 di-hydroxyalkyl, alkoxyalkyl,
(alkylthio)hydroxyalkyl, cycloalkyl, cycloalkylalkyl, hydroxy cycloalkyl, (alky 1th io)(alkylester)alkyl,
alkylesteralkyl, 2,4-dimethoxyphenyI, 3,5-trifluoromethylphenyl, 3-chlorophenyl, 4-chlorophenyl
2,6-dichlorophenyl, 2-methylphenyl, 3-methylphenyl, (substituted phenyl)alkyl, phenylalkyl,
heterocyclylalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl, optionally substituted heterocyclyl,
10 and optionally substituted heterocyclylalkyl.
5. A compound according to claim 4, racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein R.1 is hydrogen and is hydrogen.
6. A compound according to claim 4, racemic-diastereomeric mixtures thereof, optical
15 isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein
W is NO2;
Q is hydrogen;
Rl is in the 7-position and is hydrogen, methyl, ethyl or phenyl;
20 are each hydrogen;
X* is hydrogen; and
R3 is selected from the group consisting of ethyl, n-Bu, r-Bu, n-Pr, allyl, cyclopropyl, cyclobutyl,
2,4-dimethoxyphenyl, 3,5-bis-trifluoromethylphenyl, 3-chlorophenyi, 4-chlorophenyl, 2,6-
dichlorophenyl, 2-methylphenyl and 3-methylphenyl.
25 7. A compound according to claim 3, racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein
Q is H;
WisN0 2 ;
30 YisS;
Rl is in the 7-position and is hydrogen, -CH2-SC>2-phenyl, -CH 2 -CN, ~CH(CH 3 )(CN), or -
CH(CN)(CH 2 -phenyl);
R2 is hydrogen;
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Xl is hydrogen, methyl or -(CH2)2-OH;
R- 5 is selected from the group consisting of ethyl, benzyl, EtOH, n-PrOH, n-BuOH, n-pentanol, n-
hexanol, -(CH2)2-NH-(CH2>2-OH, -(CH2)2-0-(CH2)2-OH, -CH(CH 2 CH3)(CH 2 OH),
-CH(CH 2 OH)(CH 2 -/-Pr), 2,3-di-hydroxy-propyI, 2-hydroxypropyl, -CH(CH 3 )(CH 2 OH),
-C(CH3) 2 (CH 2 OH), -CH2(CH3)(CH 2 OCH 3 ), 1,3-dihydroxyisopropyl, -
CH(CH20H)(CH2CH2SCH3), cyclopropyl, cyclopropylmethyl, 4-hydroxycyclohexyl, 3-
chlorophenyl, 4-chlorophenyl, 2-methylphenyl, 3-methylphenyl, 4-aminobenzyl, (4-
aminophenyI)ethyl, ~(CH2)3-N(Et)2, -(CH2)2-N(Me)2, N-piperidinyl, 2,6-dimethylpiperidinyl,
-(CH 2 ) 2 -N \ -CH 2 -Y N - CH 2— ~^J? _CH2 "V_) -( CH 2)3— N O
, , ' and ^ .
10 8. A compound according to claim 3, racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein
* Y is O;
R 1 is in the 7-position and is hydrogen, -CH2-S02-phenyl, -CH2-CN, -CH(CH3)(CN), or
1 5 -CH(CN)(CH 2 -phenyl);
R2 is hydrogen;
is hydrogen, methyl or ~-(CH2)2-OH;
R 3 is selected from the group consisting of benzyl, EtOH, n-PrOH, /-BuOH, n-hexanol, aminoethyl,
aminopropyl, -(CH 2 )2-NH-(CH2)2-OH, -(CH 2 )2-0-(CH 2 )2-OH, -CH(CH 2 CH 3 )(CH20H),
20 -CH(CH20H)(CH 2 -/-Pr), 2,3-di-hydroxy-propyl, 2-hydroxypropyl, -CH(CH3)(CH 2 OH), 1 ,3-
dihydroxyisopropyl, -CH(CH20H)(CH2CH2SCH3), cyclobutyl, 4-hydroxycyclohexyl,
-CH(COOEt)(CH 2 )2-SCH 35 -(CH 2 )2-COOEt, -(CH 2 )5-COOEt, (2~aminophenyI)methyI, 4-
aminobenzyl, (4-aminopheny])ethyl, -C(CH3) 2 (phenyl), -CH 2 (2,4-difluorophenyl), 2-
pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl -(CH 2 )2-thien-2-yl, -CH(/-Pr)(COOEt), -CH(/-
25 Pr)(CH 2 OH), 3-(N-methyIamino)propyl, -(CH 2 )3-N(Et) 2? -(CH 2 )4-N(Et) 2 , -CH(Me)(CH 2 )4-CH3,
-CH(Me)(CH 2 )3-N(Et)2 9 N-piperidinyl, -(CH 2 )2-(4-(S02NH 2 )phenyl), 2,6-dimethylpiperidinyl,
O
N—COOEt -(CH 2 ) 2 — J
7 and ^
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9. A compound according to claim 3, racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein
W is NO2;
Q is hydrogen;
Rj is in the 7-position and is -CH2-C0 2 -t-Bu, allyl or benzyl;
R2 are each hydrogen;
is hydrogen; and
R3 is ethyl.
1 0. A compound according to claim 3, racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein
W is NO2;
Rl is in the 7-position and is hydrogen, -CH(CH 3 )(CN) or -CH(CN)(CH 2 -phenyl);
15 R2 is hydrogen; and
Y
10
— R 3
N
Q is taken together with X 1 and to form , where Y is O and R3 is ethyl.
11. A compound according to claim 2, racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein
20 W is N0 2 ;
Qis H;
Rl and R 2 are each hydrogen; and
R3 and X 1 are taken together with the nitrogen atom to which they are attached to form
/ \
-N N-COO-t-Bu
\ /
or
25 1 2. A compound according to claim 3, racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein
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15
20
W is NO2;
Rl is hydrogen or is in the 7-position and is -CH2-CN, -CH2-CONH2 and ~CH2-COO-t-Bu;
is hydrogen;
X* is hydrogen or ---CH2-O-CH3;
R3 is methyl, ethyl, n-BuOH, -CH2CF3, morpholino, -(CH 2 )7-N(Me)2, 2-phenyl-phenyl, n-BuOH ;
-CH 2 CF 3 , morpholino, -(CH 2 )4-N(Me) 2 , -(CH 2 )2-N(Me) 2 , -(CH 2 )3-NHMe, benzyl or-CH 2 -0-
CH 3 ;
Y
N
N— FT
N
or Q is hydrogen or is taken together with to form
ethyl;
where Y is O and R^ is
10 or R^ and X 1 are taken together with the nitrogen atom to which they are attached to form
25
/ \
-N N-Z
H or Me ? w h ere 2 i s methyl, 4-fluorophenyl, 2-pyridyl, 2-methoxyphenyl, -CH 2 -
CH=CH-phenyl or 2,4-dimethoxyphenyl.
13. A compound according to claim 1, racemic-diastereomeric mixtures thereof, optical
isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said
compound, isomers, prodrugs and isotopes, wherein
W is CI or Br;
Qis H;
R3 is an optionally substituted moiety selected from the group consisting of alkyl, alkenyl, phenyl,
phenylalkyl, heterocyclyl, heterocyclyl-alkyl or aminoalkyl.
14. A compound according to claim 13, racemic-diastereomeric mixtures thereof,
optical isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of
said compound, isomers, prodrugs and isotopes, wherein
R3 is alkyl, haloalkyl, esteralkyl, N,N-dialkylaminoalkyl, alkenyl, phenyl, phenylalkyl, halophenyl,
alkoxyphenyl, aryloxyphenyl, thienyl-alkyl, halopyridyl, heterocyclyl, heterocyclyl-alkyl or
aminoalkyl.
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15. A compound according to claim 14, racemic-diastereomeric mixtures thereof,
optical isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of
said compound, isomers, prodrugs and isotopes, wherein
Wis CI;
r3 is ethyl, propyl, butyl, t-butyl, 2,4,6-trichlorophenyl, 2,4-dimethoxyphenyl, -(CH2)2-2-thienyI,
allyl, 2-bromoethyl, 2-phenoxyphenyl, 2,6-dichloropyrid-4-yl, benzyl, -(CH2) 2 -COOEt, -(CH 2 )3-
N(Et) 2 , -(CH 2 )4-N(Et) 2 , or -(CH 2 ) 2 -N(Me) 2 .
16. A compound according to claim 15, racemic-diastereomeric mixtures thereof,
optical isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of
said compound, isomers, prodrugs and isotopes, wherein R 3 is -(CH2)2-2-thienyl, allyl, 2-
bromoethyl, 2-phenoxyphenyl, 2,6-dichloropyrid-4-yl, benzyl, -(CH2)2-COOEt, -(CH2>3-N(Et)2,
-(CH 2 )4-N(Et) 2 , or -(CH 2 )2-N(Me) 2 .
1 7. A compound of the formula
O
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein
R3 is ethyl, propyl, t-butyl, 2,4,6-trichlorophenyl or 2,4-dimethoxyphenyl.
18. A compound according to claim 14, racemic-diastereomeric mixtures thereof,
optical isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of
said compound, isomers, prodrugs and isotopes, wherein
r3 is hydroxy, nitro, or an optionally substituted moiety selected from the group consisting of alkyl,
alkoxy, arylalkyloxy and sulfonato;
R2 is halo or nitro; and
R3 is alkyl or phenylalkyl.
19. A compound according to claim 18, racemic-diastereomeric mixtures thereof,
optical isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of
said compound, isomers, prodrugs and isotopes, wherein
Rl is hydroxy, nitro, methyl, methoxy, isopropoxy, benzyloxy, 4-fluorobenzyloxy, -O-
C(CH 3 )2(C(0)NH 2 ), -0-(CH 2 )2-0-(CH 2 )2-OMe or-0-S02-CF 3 ;
R2 is CI or nitro; and
175
WO 01/57008 PCT/US01/03803
R.3 is ethyl or benzyl.
20. A compound according to claim 19, racemic-diastereomeric mixtures thereof,
optical isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of
said compound, isomers, prodrugs and isotopes, wherein X'isH .
5 21. A compound according to claim 20, racemic-diastereomeric mixtures thereof,
optical isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of
said compound, isomers, prodrugs and isotopes, wherein W is CI; R 1 is in the 7-position; and R 2 is
in the 4- or 5-position.
22. A compound of the formula
R 1
racemic-diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof
or pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes, wherein R*
is methyl, methoxy or isopropoxy.
23. A method of inhibiting protein kinase activity, which comprises administering to a
15 patient a compound of formula (IB) as defined hereinabove, racemic-diastereomeric mixtures
thereof, optical isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable
salts of said compound, isomers, prodrugs and isotopes.
24. The method according to claim 23, wherein said protein kinase is a tyrosine kinase.
25. The method according to claim 24, wherein said tyrosine kinase is a receptor
20 tyrosine kinase or a non-receptor tyrosine kinase.
26. The method according to claim 25, wherein tyrosine kinase is K.DR or Lck.
27. The method according to claim 23, wherein said tyrosine kinase affects
angiogenesis.
28. The method according to claim 27, wherein the inhibition of said tyrosine kinase
25 results in an anti-angiogenic effect.
29. A method of treating a condition, disorder or disease, which comprises
administering to a patient a compound of formula (IB) as defined hereinabove, racemic-
diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof or
pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes;
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where said condition, disorder or disease is selected from the group consisting of hyperproliferative
disorders, an ulcer, Lyme disease, sepsis, von Hippel Lindau disease, pemphigoid, psoriasis,
psoriasis arthropathy, paraneoplastic syndrome, turbid effusions, coilagenosis, Lupus
erythematosus, poly-myositis, dermato-myositis, systemic sclerodermia, mixed coilagenosis,
5 postinfectious arthritis, seronegative spondylarthritis, spondylitis ankylosans, vasculitis, sarcoidosis,
arthrosis, pain, Paget' s disease, polycystic kidney disease, fibrosis, sarcoidosis, cirrhosis, thyroiditis,
hyperviscosity syndrome, Osier- Weber-Rendu disease, chronic occlusive pulmonary disease,
ovarian hyperstimulation syndrome, preeclampsia, menometrorrhagia, endometriosis, chronic
inflammation, systemic lupus, glomerulonephritis, synovitis, inflammatory bowel disease, Crohn's
10 disease, glomerulonephritis, rheumatoid arthritis, juvenile arthritis, osteoarthritis, multiple sclerosis,
graft rejection, sickle cell anaemia, an ocular condition, a cardiovascular condition, atherosclerosis,
restenosis, ischemia/reperfusion injury, vascular occlusion, carotid obstructive disease, cancer,
Crow-Fukase (POEMS) syndrome, a diabetic condition, anemia, ischemia, infarct, transplant
rejection, a wound, gangrene, necrosis, asthma or edema following burns, trauma, radiation, stroke,
15 hypoxia or ischemia, and infection by Herpes simplex, Herpes Zoster, human immunodeficiency
virus, parapoxvirus, protozoa or toxoplasmosis.
30. The method according to claim 29, wherein the ocular condition is ocular or macular
edema, ocular neovascular disease, scleritis, radial keratotomy, uveitis, vitritis, myopia, optic pits,
chronic retinal detachment, post-laser treatment complications, conjunctivitis, Stargardt's disease,
20 Eales disease, retinopathy or macular degeneration.
3 1 . The method according to claim 29, wherein the cancer is a solid tumor, a sarcoma,
fibrosarcoma, osteoma, melanoma, retinoblastoma, a rhabdomyosarcoma, glioblastoma,
neuroblastoma, teratocarcinoma, an hematopoietic malignancy, malignant ascites, Kaposi's sarcoma,
Hodgkin's disease, lymphoma, myeloma or leukemia.
25 32. The method according to claim 29, wherein the diabetic condition is insulin-
dependent diabetes mellitus glaucoma, diabetic retinopathy or microangiopathy.
33. A method of decreasing fertility in a patient, which comprises administering to a
patient an effective amount of a compound of formula (IB) as defined hereinabove, racemic-
• diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof or
30 pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes.
34. A method of promoting angiogenesis or vasculogenesis, which comprises
administering to a patient a compound of formula (IB) as defined hereinabove, racemic-
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10
diastereomeric mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof or
pharmaceutically-acceptable salts of said compound, isomers, prodrugs and isotopes.
35. A method according to claim 34, wherein the compound of formula (IB) is
administered in combination with a pro-angiogenic growth factor.
36. A method of treating a patient having a condition which is mediated by protein
kinase activity, said method comprising the step of administering to the patient a therapeutically
effective amount of a compound of formula (IB) as defined hereinabove, racemic-diastereomeric
mixtures thereof, optical isomers thereof, prodrugs thereof, isotopes thereof or pharmaceutically-
acceptable salts of said compound, isomers, prodrugs and isotopes.
37. The method according to claim 36, wherein the protein kinase activity is involved in
T cell activation, B cell activation, mast cell degranulation, monocyte activation, the potentiation of
an inflammatory response or a combination thereof.
38. A pharmaceutical composition comprising a compound according to claim 1 and a
pharmaceutically acceptable diluent or carrier.
15 39. A pharmaceutical composition for inhibiting a protein kinase, which composition
comprises a pharmaceutically acceptable carrier or diluent and an effective amount of a compound
of formula (IB) as defined hereinabove, racemic-diastereomeric mixtures thereof, optical isomers
thereof, prodrugs thereof, isotopes thereof or pharmaceutically-acceptable salts of said compound,
isomers, prodrugs and isotopes.
20 40. A compound according to claim 1, wherein W is -(CH2)2-NH-C(O)-NH-(C(R 10 )2) a -Z 1 q or
an optionally substituted heterocyclyl; Ri and R 2 are each H; Q is H; Y is O; X* is H; and R 3 is an
optionally substituted alkyl.
41. A compound according to claim 40 wherein W is:
25 -(CH 2 )2-NH-C(0)-NH-Et, -CH 2 -NH-C(0)-NH-ethyl, -CH 2 -NH 2 , -NH-phenyl, -C(O)-
NH 2 , -CH 2 -NH-S(0) 2 -Ph ? -C(0)-NH-phenyl, -CH 2 -NH-S(0) 2 -CF 3 , -CH 2 -CN, -CH 2 -NH-
CH 2 -5-methyl-furan-2-yl ? -C(0)-NH-(CH 2 ) 3 -(4-methylpiperazin- 1 -yl),
-(CH 2 )2-NH-C(0)-NH-(phenyl) s or -(CH 2 )2-NH-C(0)-NH-(p-toluyl).
178
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PCT/US01/03803
42. A compound according to claim 41, wherein R J is ethyl.
43. A compound according to claim 1, wherein W is CN; R 1 and R 2 are each H; Q is H; Y is O;
X 1 is H; and R 3 is an optionally substituted heterocyclyl-heterocyclyl, or heterocyclyl-
cycloalkyl.
5 44. A compound according to claim 1, wherein R 3 is 3-(4-methylpiperazino)propyl, 2-
morpholinoethyl, 3-(9-benzyI-9-azabicycIo[3.3.1]nonyl, 6-(4-methylpiperazino)-3-pyridyl, 3-(8-
benzyl-8-azabicyclo[3.2.1]octyl, methyl-3-(8-benzyl-8-azabicyclo[3.2.1]octyl 5 /er/-butylcarboxylate-
l-piperidinylmethyl ? 4-piperidylmethyl, /e^butylcarboxylate-l-piperazinyl-ethyl, 2-piperazinoethyl,
4-(4-methylpiperazino)cyclohexyl, 3-piperidinopropyl, 6-(4-methylpiperazino)-3-pyridyl.
10 45. A compound according to claim 1, wherein R 1 and W are taken together to form
where is independently selected from the same group of substituents as X J .
46. A compound according to claim 45, wherein R 2 is H; Q is H; Y is O; X* is H; R 3 is alkyl;
15 and X 10 is ethyl, 3-pyridyl, N-(p-Br-phenyl)-NH-, 1-piperidyl or CH3-NH-.
47. A compound according to claim 1, wherein W is H; and R 1 is -S-X 3 , -S(0)X 3 or -
S(0) 2 X 3 .
48. A compound according to claim 1, wherein W is Br, CI or p-fluorophenoxy, R 1 and R 2 are
each H; Q is H; Y is O; X 1 is H; and R 3 is alkyl-chloro,
20
179
WO 01/57008
PCT/US01/03803
-alkyl
-alkyl
-alkyl-piperazin-l-yl, -alkyl-(2 3 5-dimethylpiperazin-l-yl) ? -alky]-(3,5-dimethylpiperazin-l-yl), -
alkyl-(3-aminocarbonylpiperidin-l-yl), -alkyl-(4-hydroxypiperidin-l-yl), -alkyl-(3-
5 hydroxypiperidin-l-yl), -alkyl-COOEt, -alkyl-COOH, -alkyl-(4-methylpiperazin-l-yl), -alkyl-
(N-morpholinoethylamino), -alkyl-(N-piperidinylethylamino), -aIkyl-(N-(N,N-
diethylaminoethyl)-N-(methyl)amino) ? -alkyl-((l-ethylpyrrolidin-2-yl)-methylamino) 9 -alkyl-
(N-(l-methylpiperidin-4»yI)-N-(methyl)amino) 5 -alkylamino, -alkyl-piperidin-l-yl or -alkyl-
(N,N-diethylaminoethylamino).
10 49. A compound according to claim 48, wherein the alkyl group is methylene, ethylene or
propylene.
50. A compound according to claim 1, wherein R 2 is H; Q is H; Y is O; X 1 is H and R3 is ethyl.
51. A compound according to claim 50, wherein W is H or Br; and R 1 is in the 7-position of the
benzothiazolyl ring and is -OCH, -C^C-(2-pyridinyl), -C^C-CH 2 -N(CH 3 ) 25 -0-CH(CH 3 ) 2?
1 5 phenyl or -CH-CH2.
52. A compound according to claim 50, wherein R 1 is -CH=CH2 and W is -CH=CH2.
53. A compound according to claim 50, wherein R 1 is H and W is benzyl, p-fluorophenoxy or
pyridin-4-ylmethyl.
54. A compound according to claim 50, wherein W is F; R 1 is in the 7-position of the
20 benzothiazolyl ring and is H or CI; and R 2 is in the 5-position of the benzothiazolyl ring and is H or
CI.
55. A compound according to claim 50, wherein R 1 is H and W is -CtfeCH, -C-C-Ph, -C=C-
CH 2 -N(CH 3 ) 2 , -C^C-(4-fluorophenyl), -CsC-(p-toIuyl), -(CH 2 ) 2 -Ph, ~(CH 2 ) 2 -(4-fluorophenyl), -
CH=CH-phenyl, -CH=CH-CH 2 -N(CH 3 ) 2 , ~CH=CH-(4-fluorophenyl), -CH=CH-(p-toluyl), or-
25 CH=CH-(l-imidazolyl).
56. A compound according to claim 1, wherein W is p-fluorophenoxy, -(CH2)3-NHMe or-
(CH 2 )2-l"Piperazinyl; and is ^H 2 -C(Me)2-CH 2 -N(CH 3 ) 2 , -(CH 2 ) 2 -(5-imidazolyl),
180
WO 01/57008
PCT/US01/03803
-<CH 2 ) 3
-CH
-CH
2
\
/
, or
57. A compound according to claim 1, wherein R 1 is in the 7-position of the benzothiazolyl rin
and is H or CN; R 2 is H; Y is O; Q and X 1 are each H;
W is CI, N0 2 , -CH 2 -OH, -CH 2 -0-C(0)-NH-Et, -S-phenyl, -O-phenyl, -S-CH3, -C(0)-phenyl, -
S(0)-phenyl, -S-/?-nitrophenyl, -S-p-methylphenyl, -S-p-chlorophenyl, -S-j>methoxyphenyl, -S-m-
CF 3 -phenyl, -S-o-chlorophenyl, -C(0)-CH 3 , -NH-C(0)-NH-(-CH 2 ) 2 -2-thienyl, -NH-C(0)-NH-3-
pyridyl, -S(0)2-p-(carboxymethylamino)-phenyl, -N-morpholino, -NH-C(0)-NH-Et, -NH-C(O)-
NH-CH2-phenyl, -S-p-chlorophenyl, -S-^-bromophenyl, -S-w-CF3-phenyl, or -S-/?-fluorophenyl;
R J is
Ph
{/ \\ — N N— Me
\ /
-CH.
O
J °
, ethyl, -(CH2)3-4-methylpiperazin-l-yl, -
(CH2)2- N - mor R h °l ino > or -CH2-piperidin-4-yl.
58. A compound of the formula
181
WO 01/57008
PCT/US01/03803
wherein W is H, -OCF 3 , -O-Et, F, CH 3 , -OCH 3 , -S0 2 -Me, NH 2 , -NH-C(0)-Me, -NH-CH 2 -phenyI,
-NH-S(0) 2 -2-thienyl, -NH-S(0) 2 <3,5-dimethyIisoxazol-4-yl), -NH-S(0) 2 -Me, -NH-S(0) 2 -CH 2 -
phenyl, -NH-C(0)-0~CH 2 -CC1 3 , -NH-C(0)-0-CH 2 -Ph, -NH-C(0)-0-Me or N0 2 ;
Rl is H, F or -CH 2 -S(0) 2 -phenyl; and
5 R 2 is H, 4-Cl 5 4-methyl, 5-^methyl, 5-C1, 5-F or 5-OCH 3 .
59. A method of using a compound of formula (IB) or a pharmaceutical ly acceptable
salt thereof as a replacement therapy for anti-inflammatory glucocorticosteroid therapy in a patient
undergoing anti-inflammatory glucocorticosteroid therapy comprising the step of replacing a
glucocorticosteroid with a compound of formula (IB) or a pharmaceutical^ acceptable salt thereof.
0 60. A method of using a compound of formula (IB) or a pharmaceutical ly acceptable
salt thereof in conjunction with glucocorticosteroid therapy in a patient undergoing
glucocorticosteroid therapy comprising the step of replacing a portion of the amount of
glucocorticosteroid administered to said patient.
182
INTERNATIONAL SEARCH REPORT
Int tional Application No
PCT/US 01/03803
A. CLASSIFICATION OF SUBJECT MATTER
IPC 7 C07D277/82 C07D417/04
C07D451/02 C07D451/14
A61K31/435 A61K31/53
C07D417/12
C07D513/04
A61P35/00
C07D417/06 C07D453/02
C07D487/08 A61K31/428
According to international Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
IPC 7 C07D A61K A61P
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practical, search terms used)
EPO-Internal, WPI Data, BEILSTEIN Data, CHEM ABS Data
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category °
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
x
WO 99 00357 A (VERTEX PHARMACEUTICALS
INCORPORATED) 7 January 1999 (1999-01-07)
the whole document
WO 98 04536 A (OTSUKA PHARMACEUTICAL
COMPANY, LIMITED)
5 February 1998 (1998-02-05)
the whole document
WO 99 24035 A (BRISTOL-MYERS SQUIBB
COMPANY) 20 May 1999 (1999-05-20)
the whole document
-/--
1-60
1-60
1-60
LH
Further documents are listed in the continuation of box C.
ID
Patent family members are listed in annex.
° Special categories of cited documents :
"A" document defining the general state of the art which is not
considered to be of particular relevance
"E" earlier document but published on or after the international
filing date
■L 1 document which may throw doubts on priority claimfs) or
which is cited to establish the publication date of another
citation or other special reason (as specified)
"O" document referring to an oral disclosure, use, exhibition or
other means
"P" document published prior to the international filing date but
later than the priority date claimed
T" later document published after the international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention
"X" document of particular relevance; the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document is taken alone
"V document of particular relevance; the claimed invention
cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.
document member of the same patent family
Date of the actual completion of the international search
21 May 2001
Date of mailing of the international search report
11/06/2001
Name and mailing address of the ISA
European Patent Office, P.B. 5818 Patentlaan 2
NL-2280 HV Rijswyk
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl,
Fax: (+31-70) 340-3016
Authorized officer
Allard, M
Form PCT/ISA/210 (second sheet) (July 1992)
INTERNATIONAL SEARCH REPORT
1 atlonal Application No
PCT/US 01/03803
C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT
Category °
Citation of document, with indication .where appropriate, of the relevant passages
Relevant to claim No.
X
DATABASE WPI
1-60
Section Ch, Week 199945
Derwent Publications Ltd., London, GB;
Class B03, AN 1999-530743
XP002167835
& JP 11 222431 A (OTSUKA PHARM CO LTD),
17 August 1999 (1999-08-17)
abstract
X
DATABASE WPI
1-60
Section Ch, Week 199930
Derwent Publications Ltd., London, GB;
Class B02 AN 1999-352846
XP002167836
& JP 11 130761 A (OTSUKA PHARM CO LTD),
18 May 1999 (1999-05-18)
abstract
P,X
WO 01 07411 A (BANYU PHARMACEUTICAL CO.,
1-60
LTD.) 1 February 2001 (2001-02-01)
the whole document
X
US 3 725 428 A ( JANIAK S)
17,58
3 April 1973 (1973-04-03)
the whole document, particularly compounds
12, 14 and 15
X
— — —
GB 1 580 876 A (NIHON T0KUSHU NOYAKU SEIZ0
1-3
K.K.) 10 December 1980 (1980-12-10)
the whole document
X
F W BELL: "Phenethyl thi azol ethi ourea
1— o , So ,
(PETT) comDounds . a new class of HIV-1
39
reverse transcriptase inhibitors.
Synthesis and basic structure-activity
relationship studies of PETT analogs"
JOURNAL OF MEDICINAL CHEMISTRY,
vol. 38, no. 25, 1995, pages 4929-4936,
XP002108199
the whole document, particularly page
4931, table 4, compound 54
X
DATABASE CAPLUS 'Online!
1
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1970:55311
XP002167822
RN 25048-85-5 and 25048-86-6
& INDIAN 0. APPL. CHEM. ,
vol. 31, no. 3-4, 1968, pages 117-120,
Form PCT/ISA/210 (continuation of second sheet) (July 1992)
INTERNATIONAL SEARCH REPORT
Ini ional Application No
PCT/US 01/03803
C.(Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT
Category °
Citation of document, with indication .where appropriate, of the relevant passages
Relevant to claim No.
X
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1972:14418
XP002167823
RN 34990-07-3 and 34990-08-4
& CURR. SCI.,
vol. 40, no. 6, 1971, pages 430-432,
l
x
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1974:82772
XP002167824
RN 51335-50-3, 51335-51-4, 51335-52-5,
51335-57-0 and 51335-58-1
& CHEM. ZVESTI,
vol. 27, no. 6, 1973, pages 829-833,
1,38,39
X
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1974:108419
XP002167825
RN 1819-39-2 and 1819-47-2
& CURR. SCI. ,
vol. 43, no. 2, 1974, pages 33-36,
1
X
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1974:116995
XP002167826
RN 18704-01-3
& CURR. SCI.,
vol. 42, no. 20, 1973, pages 717-718,
1
X
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1980:22425
XP002167827
RN 1819-37-0, 1819-43-8 and 1819-50-7
& J. INDIAN CHEM. SOC,
vol. 56, no. 4, 1979, pages 377-380,
1
X
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1992:165134
XP002167828
RN 1819-50-7 and 1819-51-8
& J. INDIAN CHEM. SOC. ,
vol. 68, no. 9, 1991, pages 517-518,
' _/--
1
Form PCT/1SA/210 (continuation of second sheet) (July 1992)
INTERNATIONAL SEARCH REPORT
fruw^,. u tional Application No
PCT/US 01/03803
C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT
Category
x
x
x
Citation of document, with indication .where appropriate, of the relevant passages
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1994:95413
XP002167829
RN 152266-96-1
& RES. COMMUN. PSYCHOL., PSYCHATRY BEHAV. ,
vol. 17, no. 3-4, 1992, pages 153-159,
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1991:134832
XP002167830
RN 21718-27-4 and 132629-14-2
& 0. IRAQI CHEM. SOC. ,
vol. 13, no. 1, 1988, pages 103-115,
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1991:23848
XP002167831
RN 21718-27-4, 131120-20-2, 131120-21-3
and 131120-22-4
& INDIAN J. CHEM. , SECT. B,
vol. 29B, no. 8, 1990, pages 778-780,
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS ,
OHIO, US;
Database accession no. 1988:429972
XP002167832
RN 111962-93-7
& JP 62 187842 A (KONICA CO.)
17 August 1987 (1987-08-17)
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1983:198237
XP002167833
RN 84426-92-6, 84426-99-3, 84427-14-5 or
84427-15-6
& OP 57 175189 A (KYOWA HAKKO KOGYO CO. ,
LTD.) 28 October 1982 (1982-10-28)
DATABASE CAPLUS 'Online!
CHEMICAL ABSTRACTS SERVICE, COLUMBUS,
OHIO, US;
Database accession no. 1995:885379
XP002167834
RN 56159-92-3, 56159-94-5 or 172750-38-8
& ASIAN J. CHEM. ,
vol. 7, no. 4, 1995, pages 832-836,
Relevant to claim No.
1,38,39
1
1,38,39
Form PCT/ISA/210 (continuation of second shsei) (July 1992)
INTERNATIONAL SEARCH REPORT
International Application No. PCTAJS 01 £3803
FURTHER INFORMATION CONTINUED FROM PCT/iSA/ 210
Continuation of Box 1.2
Claims Nos.: 1-3, 8, 38-42, 44-60 (all partly searched)
The initial phase of the search revealed a very large number of documents
relevant to the issue of novelty. So many documents were retrieved that
it is impossible to determine which parts of the claim(s) may be said to
define subject-matter for which protection might legitimately be sought
(Article 6 PCT).
For these reasons it appears impossible to execute a meaningful search
and/or to issue a complete search report over the whole breadth of the
above mentioned claim(s).
The search and the report for those claims can only be considered
complete for compounds of formula (I) according to claim 1 wherein W is
CI , Br, N02 or CN.
The applicant's attention is drawn to the fact that claims, or parts of
claims, relating to inventions in respect of which no international
search report has been established need not be the subject of an
international preliminary examination (Rule 66.1(e) PCT). The applicant
is advised that the EPO policy when acting as an International
Preliminary Examining Authority is normally not to carry out a
preliminary examination on matter which has not been searched. This is
the case irrespective of whether or not the claims are amended following
receipt of the search report or during any Chapter II procedure.
INTERNATIONAL SEARCH REPORT
Information on patent famify members
Im&rorcicional Application No
PCT/US 01/03803
Patent document
Publication
Patent family
Publication
cited in search report
date
member(s)
date
IJA QQAAQC7
A
f»7— H1 —1 OQQ
u/ u i xyyy
US
6093742
a
ri
AU
8377698
a
n
iq_ni-iQQQ
X 27 UX 1733
EP
0993441
ft
r\
X-7 U^r C.VJVJU
WO 9804536
A
05-02-1998
AU
695817
B
20-08-1998
AU
3635497
a
CA
2233611
A
VJO Ul X-7-70
CN
1198160
A
EP
0858452
A
1Q-0R-1QQ8
JP
10095777
A
r\
X *r U4 X -7 O
US
6140330
A
OX lu C.UUU
WO 9924035
A
20-05-1999
AU
1371999
A
31-05-1999
BR
9814956
A
03-10-2000
CN
1290165
T
04-04-2001
EP
1037632
A
27-09-2000
NO
20002121
A
09-05-2000
PL
340727
A
26-02-2001
TR ,
200001312
T
21-09-2000
TP 11 OOOA *i 1
Or ILl.l.c.QoL
A
n
± /— uo
NONE
ID 111 Qf\"7A1
A
Pi
1 Q-HR-I QQQ
NONE
WU UiU/411
A
Ul _ U£—
NONE
A
UO U^f lr / J
CH
505543
A
r\
1 R-04-1 Q71
1 D UH X -7 / X
AT
295922
R
D
ID X J / X
BE
741093
A
rt
JU U*T 1 U
BG
17439
A
r\
i 0-1 1 -1Q7^
XV/ XX X 27 / *->
526256
A
r\
UO X -7 / £.
cs
164834
D
D
lO XX 15/ j
DE
1953149
A
06-05-1970
DK
128742
B
24-06-1974
FR
2022369
A
r\
GB
1290388
A
M
97-0Q-1Q7?
IL
33221
A
oq-0fi-1Q7^
NL
6916457
A
M
0fi-0R-1Q7D
UL> Uj Ij / u
RO
61385
A
X 3 X £_ X -7 / U
IIS
3810988
A
1 4-0R-1 Q74
X *t X -7 I f
GB 1580876
A
10-12-1980
JP
53124265
A
30-10-1978
RF
865757
A
06-10-1978
BR
7802111
a
DE
2813826
A
19-10-1978
L/I\
151278
X»J X C f \J
A
07-10-1978
FR
2386532
U <J XX X 27 / O
IT
1094316
B
26-07-1985
NL
7803624
A
10-10-1978
JP 62187842
A
17-08-1987
JP
1838541
C
25-04-1994
JP
5049088
B
23-07-1993
JP 57175189
A
28-10-1982
JP
1482183
C
27-02-1989
JP
63032073
B
28-06-1988
Form PCT/lSA/210 (patent family annex) (July 1992)
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